Protein Gel Electrophoresis

Western blotting
Protein gel electrophoresis

technical handbook
separate transfer d
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Comprehensive solutions
designed to drive your success
Protein gel electrophoresis is a simple way to
separate proteins prior to downstream detection
or analysis, and is a critical step in most
workflows that isolate, identify, and characterize
proteins. We offer a complete array of products
to support rapid, reliable protein electrophoresis
for a variety of applications, whether it is the
first or last step in your workflow. Our portfolio
of high-quality protein electrophoresis products
unites gels, stains, molecular weight markers,
running buffers, and blotting products for
your experiments.
For a complete listing of all available products

and more, visit thermofisher.com/separate
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Prepare samples Choose the electrophoresis
Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain 3
Contents
Electrophoresis overview 4
Select precast gel

Gel selection guide 8
Gels 10
Prepare samples and select buffers

Sample prep kits 26
Buffers and reagents 28
Buffers and reagents selection guide 29
Select the standard

Protein ladders 34
Protein standards selection guide 36
Choose the electrophoresis chamber system and power supply

Electrophoresis chamber systems 50
Electrophoresis chamber system selection guide 51
Power supplies 58
Run the gel

Gel run conditions 59
Troubleshooting tips 60
Stain the gel

Protein stains 62
Protein stains selection guides 63, 67, 69, 70
Electrophoretic staining technology 71
Post stain
Transfer and detection 74
Appendix
Protocol quick reference 76
Ordering information 81
Sample preparation and Electrophoresis chamber Electrophoresis

Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
4
Electrophoresis
Electrophoresis is defined as the Support matrix
transport of charged molecules Two types of support matrices are commonly used in
electrophoresis—polyacrylamide and agarose. The support
through a solvent by an electric matrices act as porous media and behave like a molecular sieve.
Separation of molecules is dependent upon the gel pore size of
field. Electrophoresis is a simple, the support matrix used. Agarose has a large pore size and is ideal
for separating macromolecules such as nucleic acids and protein
rapid, and sensitive analytical complexes. Polyacrylamide has a smaller pore size and is ideal for
separating most proteins and smaller nucleic acids.
tool for separating proteins and
Polyacrylamide gel electrophoresis
nucleic acids. Any charged ion or
(PAGE)
molecule will migrate when placed Polyacrylamide gels are generated by the polymerization of
in an electric field. Most biological acrylamide monomers. These monomers are crosslinked into
long chains by the addition of bifunctional compounds such as
molecules carry a net charge at any N,N,-methylenebisacrylamide (bis), which react with the free
functional groups of the chain termini. The concentration of
pH other than at their isoelectric acrylamide and bisacrylamide determines the pore size of the gel.
The higher the acrylamide concentration, the smaller the pore size,
point and will migrate at a rate resulting in resolution of lower molecular weight molecules and
vice versa.
proportional to their charge density. PAGE allows one to separate proteins for different applications
based on:
The mobility of a biological molecule • The acrylamide matrix
through an electric field will depend • Buffer systems
on the following factors: • Electrophoresis conditions
• Field strength
• Net charge on the molecule
• Size and shape of the molecule
• Ionic strength
• Properties of the matrix through which
the molecules migrate (e.g., viscosity,
pore size) Mini Gel Tank
The acrylamide matrix Electrophoresis conditions

The separation of molecules is dependent on the electrophoresis
Linear vs. gradient gels
conditions. Electrophoresis can be performed under the
Gels that have a single acrylamide percentage are referred to as
following conditions:
linear gels, and those with a range are referred to as gradient
gels. The advantage of using a gradient gel is that it allows the
separation of a broader range of proteins than a linear gel. Denaturing conditions
Electrophoresis is performed under denaturing conditions using
an anionic detergent such as sodium dodecylsulfate (SDS).
Continuous vs. discontinuous gels
SDS denatures and unfolds the protein by wrapping around the
Researchers occasionally refer to gels as continuous or
hydrophobic portions. SDS binds at a ratio of ~1.4 g SDS per
discontinuous. A continuous gel is a gel that has been formed
gram of protein. The resultant SDS-protein complexes are highly
from a single acrylamide solution in the entire gel cassette.
negatively charged and are resolved in the gel based on their size.
A discontinuous gel is formed from two acrylamide solutions, a
small, low-percentage stacking gel where the protein wells reside,
and a larger portion of gel that separates the proteins. In the Nondenaturing (native) conditions
traditional Tris-glycine protein gel system, the proteins are stacked Electrophoresis is performed under nondenaturing (native)
in the stacking gel between the highly mobile leading chloride ions conditions using buffer systems that maintain the native protein
(in the gel buffer) and the slower, trailing glycine ions (in the running conformation, subunit interaction, and biological activity. During
buffer). The reason for using the stacking gel is to improve the native electrophoresis, proteins are separated based on their
resolution of the bands in the gel. These stacked protein bands charge to mass ratios.
undergo sieving once they reach the separating gel.
Reducing conditions
Mini vs. midi protein gels Electrophoresis is performed under reducing conditions using
Commercial gels are available in two size formats, minigels and reducing agents such as dithiothreitol (DTT), β-mercaptoethanol
midigels. Both gels have similar run lengths, but midigels are wider (β-ME) or tris(2-carboxyethyl)phosphine (TCEP).
than minigels, allowing midigels to have more wells or larger wells.
The reducing agents completely unfold the denatured proteins
The additional wells in the midigels permit more samples or large
into their subunits by cleaving the disulfide bonds between
sample volumes to be loaded onto one gel.
cysteine residues.
Buffer systems
Electrophoresis is performed using continuous or discontinuous
buffer systems. A continuous buffer system utilizes only one
buffer in the gel and running buffer. A discontinuous buffer system
utilizes a different gel buffer and running buffer1. This system may
also use two gel layers of different pore sizes and different buffer
composition (the stacking and separating gel). Electrophoresis
using a discontinuous buffer system results in concentration of the
sample and higher resolution.
Did you know?
Arne Tiselius won the
Nobel Prize in Chemistry
Reference for electrophoretic analysis
1. Ornstein L (1964) Disc electrophoresis. 1. Background and theory. Ann N Y Acad Sci 121:321-349. of serum proteins in 1948.

6
With the Tris-acetate system (Figure 3), three ions are

Comparison of primarily involved:
• Acetate (–), the leading ion from the gel buffer
discontinuous buffer • Tricine (–), the trailing ion from the running buffer
• Tris (+), the common ion (in both gel and running buffer)
systems
This system also operates at a significantly lower pH than the Tris-
SDS-PAGE utilizes a discontinuous buffer system to concentrate glycine system, resulting in less gel-induced protein modifications.
or “stack” samples into a very sharp zone in the stacking gel at the
beginning of the run. In a discontinuous buffer system, the primary The diagrams below (Figures 1, 2, and 3) summarize the migration
anion in the gel is different (or discontinuous) from the primary differences in the stacking gel of each system.
anion in the running buffer. Both the Invitrogen™ NuPAGE™ systems
(Bis-Tris and Tris-acetate gels) and the Laemmli (Tris-glycine)
system are examples of discontinuous buffer systems and work
in a similar fashion. However, the NuPAGE system operates at a
lower pH as a result of the proprietary ions that are in the system. GLYCINE Figure 1. The Tris-glycine gel system.
(Trailing Ion)
• Gel buffer ions are Tris and chloride
PROTEIN/SDS COMPLEX
(pH 8.7)
In a Tris-glycine system (Figure 1), three ions are primarily involved: (Stacked Proteins)
• Running buffer ions are Tris, glycine,
CHLORIDE
• Chloride (–), supplied by the gel buffer, serves as the leading (Leading Ion) and SDS (pH 8.3)
ion because it has the highest attraction to the anode relative to PROGRESSION OF RUN
• Gel operating pH is 9.5
other anions in the system. Common Ion is Tris,
present in the gel and running buffers
• Glycine (–), the primary anion provided by the running buffer,
serves as the trailing ion, because it is only partially negatively
charged and remains behind the more highly charged chloride Figure 2. The Bis-Tris gel system.
MES or MOPS
ions in a charged environment. (Trailing Ion) • Gel buffer ions are Bis-Tris and
• Tris base (+), is a common ion present in both the gel and the PROTEIN/SDS COMPLEX chloride (pH 6.4)
(Stacked Proteins)
• Running buffer ions are Tris, MES or
running buffers. During electrophoresis, the gel and buffer ions CHLORIDE
(Leading Ion)
MOPS, and SDS (pH 7.3)
in the Tris-glycine system form an operating pH of 9.5 in the • Gel operating pH is 7.0
separating region of the gel. PROGRESSION OF RUN
Common Ion is Bis-tris,
present in the gel
In the case of the Bis-Tris system (Figure 2), three ions are
primarily involved:
• Chloride (–) supplied by the gel buffer, serves as the fast-moving Figure 3. The Tris-acetate gel system.
TRICINE
leading ion. (Trailing Ion) • Gel buffer ions are Tris and acetate
PROTEIN/SDS COMPLEX (pH 7.0)
• MES or MOPS (–) (depending on the running buffer choice) (Stacked Proteins)
• Running buffer Ions are Tris, tricine,
serves as the trailing ion. ACETATE and SDS (pH 8.3)
(Leading Ion)
∙ MES: 2-(N-morpholino) ethane sulfonic acid • Gel operating pH is 8.1
PROGRESSION OF RUN
∙ MOPS: 3-(N-morpholino) propane sulfonic acid Common Ion is Tris,
present in the gel and running buffer
• Bis-Tris (+) acts as the common ion present in the gel while
Tris (+) is provided by the running buffer.
The combination of a lower-pH gel buffer (pH 6.4) and running

buffer (pH 7.3–7.7) leads to a significantly lower operating pH
(pH 7.0) during electrophoresis, resulting in better sample integrity
and gel stability.
Select precast gel

High-performance
precast protein gels
If you are doing standard one-dimensional protein
electrophoresis, we have a broad range of solutions to
fit your research needs. Our selection of precast gels
consists of several different chemistries, well formats,
and gel sizes, so you can get the protein separation
you need for accurate downstream results.
Bolt Bis-Tris Plus gel.
Learn more at thermofisher.com/proteingels
Precast gels
Popular gel chemistries Specialty gels
• NuPAGE Bis-Tris gels • Novex Tricine gels

• NuPAGE Tris-Acetate gels • NativePAGE gels
• Bolt Bis-Tris Plus gels • Novex IEF gels
• Novex Tris-Glycine gels • Novex Zymogram gels
• E-PAGE gels
Did you know?
Over 45 years ago,
Ulrich K. Laemmli first
published SDS-PAGE as a
Casting your own gels? method for cleavage analysis
of structural proteins in
We offer preassembled empty cassettes, buffers, bacteriophage T4.
and reagents.
Learn more at
thermofisher.com/gelcastingaccessories

Prepare samples Choose the electrophoresis chamber
8 Select precast gel and select buffers Select the standard system and
chamber power
system supply
and power supply Run the gel Stain the gel Post stain
Gel selection guide Find the right gel for your research needs based on molecular weight,
downstream applications, and throughput requirements.
Denaturing separation
Molecular weight
Low molecular weight proteins High molecular weight proteins Broad-range molecular
and peptides (>2.5 kDa) (<500 kDa) weight separation
Novex NuPAGE
Tricine gels Tris-Acetate gels
Low throughput Medium or high throughput
E-PAGE 48-well
Application or 96-well gels
Coomassie dye High-sensitivity Downstream applications Large sample volume for

or silver staining western blotting requiring high protein integrity high detection sensitivity
(e.g., mass spectrometry)
NuPAGE NuPAGE NuPAGE Bolt Bis-Tris

Bis-Tris gels Bis-Tris gels Bis-Tris gels Plus gels
Bolt Bis-Tris Bolt Bis-Tris Bolt Bis-Tris

Plus gels Plus gels Plus gels
Novex
Tris-Glycine gels
Native separation
Molecular weight Isoelectric point Protease activity
1st Novex Tris-Glycine Novex Zymogram

dimension NativePAGE gels Novex IEF gels ZOOM™ IPG strips
gels with native buffers gels (casein,
blue casein, or gelatin
substrates)
2nd NuPAGE Bis-Tris Novex Tris-Glycine Novex Tris-Glycine Novex Tris-Glycine
dimension gels, 2D well gels, 2D well gels, 2D well ZOOM™ gels, IPG well
NuPAGE Bis-Tris NuPAGE Bis-Tris

gels, 2D well ZOOM gels, IPG well
Find the right mini gel using our interactive gel selection tool
at thermofisher.com/minigelselection
For ordering information refer to pages 81–87.

Protein gel electrophoresis technical handbook 9
Choose the right gel chemistry for your research needs

Bis-Tris chemistry vs. A
1 2 3 4 5 6 7 8 9 10
B
1 2 3 4 5 6 7 8 9 10
Tris-glycine chemistry
The most widely used gel system for separating

a broad range of proteins by SDS-PAGE is
the Laemmli system, which uses Tris-glycine
gels comprising a stacking gel component
Figure 4. Protein separation using (A) a Bolt Bis-Tris Plus gel and (B)
that helps focus the proteins into sharp bands
another manufacturer’s traditional Tris-glycine gel.
at the beginning of the electrophoretic run and
the resolving gel component that separates the
proteins based on size. This classic system uses Unlike traditional Tris-glycine gels, NuPAGE and Bolt gels are Bis-
a discontinuous buffer system where the pH and Tris HCI–buffered (pH 6.4) and have an operating pH of about 7.0.
ionic strength of the buffer used for running the gel
(Tris, pH 8.3) is different from the buffers used in the The neutral operating pH of the Bis-Tris systems provides the
stacking gel (Tris, pH 6.8) and resolving gel (Tris, following advantages over the Laemmli system:
pH 8.8). The highly alkaline operating pH of the
• Longer shelf life of 8–12 months due to improved gel stability
Laemmli system may cause band distortion, loss of
resolution, or artifact bands. • Improved protein stability during electrophoresis at neutral pH
enabling sharper band resolution and accurate results
The major causes of poor band resolution with the Laemmli (Moos et al. 1998)
system are: • Complete reduction of disulfides under mild heating conditions
(70°C for 10 minutes) and absence of cleavage of Asp-Pro
• Hydrolysis of polyacrylamide at the high pH of the resolving
bonds
gel, resulting in a short shelf life of 8 weeks
• Reduced state of the proteins maintained during
• Chemical alterations such as deamination and alkylation electrophoresis and blotting of the proteins when using
of proteins due to the high pH of the resolving gel Invitrogen™ NuPAGE™ Antioxidant
• Reoxidation of reduced disulfides from cysteine-containing
proteins, as the redox state of the gel is not constant
• Cleavage of Asp-Pro bonds of proteins when heated at

100°C in Laemmli sample buffer, pH 5.2
Choosing the right gel percentage Choosing a well format

In general, the size of the molecule being separated should and gel thickness
dictate the acrylamide or agarose percentage you choose. Use
a lower percentage gel to resolve larger molecules and a higher We offer most polyacrylamide gels in a choice of nine different well
percentage gel to resolve smaller ones. The exception to this rule formats (17 well, 15 well, 12 well, 10 well, 9 well, 5 well, 1 well,
is when performing isoelectric focusing. Refer to the gel migration 2D/preparative well, or IPG well). Two thicknesses (1.0 mm and
charts throughout this chapter to find the gel best suited for your 1.5 mm) are also available for popular gel types. If loading large
application. As a general rule, molecules should migrate through sample volumes (>30 μL), a thicker gel (1.5 mm) with fewer wells
about 70% of the length of the gel for the best resolution. When (e.g., 5 well) or a Bolt gel with its higher-capacity wedge wells
protein molecular weights are wide ranging, or unknown, gradient is more appropriate. When blotting, remember that proteins will
gels are usually the best choice. transfer more easily from a 1.0 mm thick gel than from a 1.5 mm
thick gel.

10 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain
Bolt Bis-Tris Plus mini gels

Neutral-pH gel system with a unique
wedge well design
Invitrogen™ Bolt™ Bis-Tris Plus gels are precast

polyacrylamide gels designed for optimal
separation of a broad molecular weight range of
proteins under denaturing conditions during gel
electrophoresis (Figure 6 and 7). These gels help Specifications
deliver consistent performance with a neutral-pH • Shelf life: ~16 months
environment to minimize protein degradation. The
unique wedge well design (Figure 5) allows loading • Average run time: 35 minutes
of up to 2x more sample volume than other precast • Separation range: 0.3–260 kDa
gels. Bolt gels are ideal for western blot transfer • Polyacrylamide concentrations: fixed 8%, 10%, and 12%;
and analysis along with any other technique where gradient 4–12%
protein integrity is crucial.
• Gel dimensions: 8 x 8 cm (1 mm thick)
Bolt Bis-Tris Plus gels offer: • Maximum sample volume per 12-well gel: ~40 μL, or two-thirds
of the sample well volume
• High sample volume capacity—wedge well design
allows detection of proteins in very dilute samples or ts acquired w
sul ith
Re
measurement of low-abundance proteins

• Preserved protein integrity—neutral-pH formulation
minimizes protein modifications
th e k
Mini Gel Tan
• Superior band quality and band volume—

Invitrogen™ Novex™ Bis-Tris Plus chemistry is designed to Figure 6. Bolt Bis-Tris Plus
gel electrophoresis. Protein
deliver sharp, straight bands with higher band volume
standards and samples were
loaded at 10 μL sample volumes
• Better protein resolution—gels are 10% longer, allowing
in a Bolt 4–12% Bis-Tris Plus
detection of more protein bands than standard mini gels Gel. Electrophoresis was
performed using the Mini Gel
• High lot-to-lot consistency—coefficient of variation (CV) Tank at 200 V (constant). Sharp,
of only 2% for Rf values (migrati
(migration) straight bands with consistent migration patterns were observed after
staining with Invitrogen™ SimplyBlue™ SafeStain. Images were acquired
using a flatbed scanner. Lane 1: Invitrogen™ SeeBlue™ Plus2 Prestained
Standard; Lane 2: 10 μg E. coli lysate; Lane 3: Invitrogen™ Mark12™
Unstained Standard (blend of 12 purified proteins); Lane 4: 40 μg HeLa cell
lysate; Lane 5: 20 μg HeLa cell lysate; Lane 6: 5 μg BSA; Lane 7:
40 μg Jurkat cell lysate; Lane 8: 5 μg GST fusion protein; Lane 9:
Invitrogen™ Novex™ Sharp Unstained Protein Standard; Lane 10: 5 μg
Figure 5. The unique wedge well design
β-galactosidase.
of Bolt B
Bis-Tris Plus gels.
Learn more at thermofisher.com/bolt
For ordering information refer to page 81. For quick reference on the protocol please refer to page 76.
Bolt Bis-Tris Plus gels

Figure 7. Bolt Bis-Tris Plus gel migration
chart. Optimal separation range is shown within
the gray areas.
“The new Bolt system is wonderful. I am

still amazed that I can run a PAGE gel in
23 minutes. The entire system is incredibly
user friendly from the Bolt precast gels with
wedged wells for ease of loading to the
Mini Gel Tank system. The bands produced
from the westerns were sharp and straight.
I would and have highly recommended this
system to anyone doing protein work.” —
Crystal M., Queen’s University,
Ontario, Canada
“For one of our projects in the lab, we
resolve proteins by electrophoresis to
determine the accumulation of ubiquitinated
proteins following treatment with a
proteasome inhibitor. When we resolved
the ubiquitinated proteins using the
Tris-glycine gels, we observed a smear.
However, when we switched to resolving
the ubiquitinated proteins using the Bolt
Bis-Tris gels, we were delightfully surprised
to observe individual protein bands in place
of the smear.” —Susan S., University of
Pennsylvania, Philadelphia, US
Did you know?

Timothy Updyke and
Sheldon Engelhorn filed a
patent for the neutral-pH Bis-Tris
gel system in 1996.
Recommended products
The Invitrogen™ Bolt™ Welcome Pack + iBlot™ 2 System offers a Thermo Scientific Pierce Power
complete protein separation and western blot solution by combining our Stainer is recommended for fast
Mini Gel Tank, Invitrogen™ Bolt™ gels and buffers, SeeBlue Plus2 Prestained Coomassie dye staining of
Standard, and Invitrogen™ iBlot™ 2 Gel Transfer Device with transfer stacks. Bolt Bis-Tris Plus Gels.
The Bolt Welcome Pack

+ iBlot 2 System.

NuPAGE gels
Revolutionary high-performance gels
referenced in >20,000 publications
The Invitrogen™ NuPAGE™ SDS-PAGE gel system

is a revolutionary high-performance polyacrylamide
gel electrophoresis system that simulates the
denaturing conditions of the traditional Laemmli
system. NuPAGE™ gels use a unique buffer
formulation to maintain a neutral operating pH Specifications
during electrophoresis, which minimizes protein • Shelf life:
modifications that can result in poor band – NuPAGE Bis-Tris gels: 16 months
resolution. – NuPAGE Tris-Acetate gels: 8 months
• Average run time: ~35 minutes

Gels are available in two formulations— Invitrogen™
NuPAGE™ Bis-Tris gels are ideal for separating • Separation range:
small to midsize proteins while Invitrogen™ – NuPAGE Bis-Tris gels: 1.5–300 kDa
NuPAGE™ Tris-Acetate gels are ideal for separating – NuPAGE Tris-Acetate gels: 30–400 kDa
large proteins (Figure 8). A gel migration chart is • Polyacrylamide concentrations:
shown in Figure 9. – NuPAGE Bis-Tris gels: fixed 8%, 10%, and 12%;
gradient 4–12%
NuPAGE gels are designed for: – NuPAGE Tris-Acetate gels: fixed 7%; gradient 3–8%
• Superior protein band resolution and stability— • Gel dimensions:

neutral-pH environment during electrophoresis minimizes – Mini: 8 x 8 cm (1 or 1.5 mm thick)
protein modifications – Midi: 8 x 13 cm (1 mm thick)
• More efficient western blot transfer—neutral pH prevents • Maximum sample volume per 10-well mini gel: 25 μL
reoxidation of reduced samples during protein transfer (1 mm thick); 37 μL (1.5 mm thick)
• Fast sample run times—typically 35–50 minutes
• Long product shelf life—stable for 8–16 months ts acquired w

sul ith
Re

Figure 8. NuPAGE Bis-Tris and Tris-Acetate

gel electrophoresis. Protein standards and

samples were loaded at 10 μL sample volumes
A. B. th e k
Mini Gel Tan in (A) Invitrogen™ NuPAGE™ 4–12% Bis-Tris and
(B) Invitrogen™ NuPAGE™ 3–8% Tris-Acetate gels.
Electrophoresis was performed using the Mini Gel
Tank at 200 V (constant). Sharp, straight bands were observed after
staining with SimplyBlue SafeStain. Images were acquired using a
flatbed scanner. (A and B) Lane 1: SeeBlue Plus2 Prestained Standard;
Lane 2: 10 μg E. coli lysate; Lane 3: Mark12 Unstained Standard
(blend of 12 purified proteins); Lane 4: 40 μg HeLa cell lysate; Lane 5:
20 μg HeLa cell lysate; Lane 6: (A) not used (B) 5 μg BSA; Lane 7:
40 μg Jurkat cell lysate; Lane 8: 5 μg GST fusion protein; Lane
9: Novex Sharp Unstained Protein Standard; Lane 10: 5 μg
β-galactosidase.
Learn more at thermofisher.com/nupage
For ordering information refer to page 81. For quick reference on the protocol please refer to page 76-77.
NuPAGE gels
A. NuPAGE Bis-Tris gels B. NuPAGE Tris-Acetate gels C. NuPAGE Tris-Acetate gels

(Denaturing separation) (Denaturing separation) (Native separation)
Figure 9. Migration patterns achieved in NuPAGE gels. For optimal gels. (B) Migration patterns of HiMark Unstained Protein Standard on
results, protein bands should migrate within the gray shaded areas. NuPAGE Tris-Acetate gels. (C) Migration pattern for Tris-acetate gel native
(A) Migration patterns of Invitrogen™ Novex™ Sharp Prestained Protein separation is for the Invitrogen™ NativeMark™ Unstained Protein Standard.
Standard or Novex Sharp Unstained Protein Standard on NuPAGE Bis-Tris
Invitrogen™ HiMark™ Unstained and Prestained Protein Standards are PageRuler, PageRuler Plus, and Spectra Prestained Protein Ladders
specifically designed for large protein analysis on NuPAGE Tris-Acetate are recommended for use with NuPAGE Bis-Tris gels for easy molecular
gels under denaturing conditions. Both standards offer a ready-to-load weight determination.
format and consist of 9 proteins with a size range of 40–500 kDa.
Visualize with Coomassie stain, silver stain, or fluorescent protein
stains after electrophoresis (see “Stain the gel”, page 62).

Novex Tris-Glycine gels

Laemmli-based precast gels for high
efficiency, reproducibility, and performance
Invitrogen™ Novex™ Tris-Glycine gels are based on Specifications

traditional Laemmli protein electrophoresis with • Shelf life: 1–2 months
minor modifications for maximum performance in
the precast format. These gels provide reproducible • Run time: ~90 minutes
separation of a wide range of proteins into well- • Separation range: 6–500 kDa
resolved bands (Figure 10). A gel migration chart is • Polyacrylamide concentrations:
shown in Figure 11. – Fixed concentrations available from 4% to 18%
– Gradient gels with ranges of 4–12%, 4–20%, 8–16%,
Novex Tris-Glycine gels are: and 10–20%
• Individually packaged for convenience • Gel dimensions:
– Mini: 8 x 8 cm (1 or 1.5 mm thick)
• Compatible with most protein standards for accurate size
– Midi: 8 x 13 cm (1 mm thick)
determination
• Maximum sample volume per well: 25 μL (1 mm thick);
• Flexible for use with native or denatured protein samples,
37 μL (1.5 mm thick)
with specially formulated buffers for each condition
ts acquired w
sul ith
Re

th e k
Mini Gel Tan
Figure 10. Novex Tris-Glycine

standards and samples were
loaded at 10 μL sample volumes
in 4–20% Tris-Glycine gels.
Electrophoresis was performed
using the Mini Gel Tank at
200 V (constant). Sharp, straight bands were observed after staining with
SimplyBlue SafeStain. Images were acquired using a flatbed scanner. Lane
1: SeeBlue Plus2 Prestained Standard; Lane 2: 10 μg E. coli lysate; Lane 3:
Mark12 Unstained Standard (blend of 12 purified proteins); Lane 4: 40 μg
HeLa cell lysate; Lane 5: 20 μg HeLa cell lysate; Lane 6: 5 μg BSA; Lane
7: 40 μg Jurkat cell lysate; Lane 8: 5 μg GST fusion protein; Lane 9: Novex
Sharp Unstained Protein Standard; Lane 10: 5 μg β-galactosidase.
Learn more at thermofisher.com/trisglycine
For ordering information refer to page 82–83. For quick reference on the protocol please refer to page 77-78.
Novex Tris-Glycine gels
A. Denaturing separation B. Native separation††
Large proteins* Mid-size proteins† Small proteins† Wide range†

(116–500 kDa) (20–250 kDa) (3–60 kDa) (6–200 kDa)
4% 6% 8% 10% 12% 14% 16% 18% 4–12% 8–16% 4–20% 10–20%
0%
500 kDa
260kDa
260 kDa
500 kDa
10%
260 kDa 160 kDa

260 kDa 260 kDa
260 kDa
290 kDa 160 kDa 260 kDa 110 kDa
20% 260 kDa 260 kDa
160 kDa 110 kDa
160 kDa
260 kDa
160 kDa 110 kDa 80 kDa 80 kDa
110 kDa
80 kDa 60 kDa
240 kDa 80 kDa 160 kDa
30% 60 kDa 60 kDa
290 kDa 40 kDa
60 kDa
160 kDa 80 kDa 50 kDa 260 kDa 110 kDa
110 kDa
40 kDa 50 kDa
50 kDa 160 kDa
40% 30 kDa 80 kDa
60 kDa
60 kDa 40 kDa
110 kDa 160 kDa
50 kDa 110 kDa
160 kDa 30 kDa
50% 50 kDa
20 kDa
80 kDa
60 kDa 110 kDa
40 kDa
40 kDa
30 kDa 15 kDa
60% 20 kDa
50 kDa 50 kDa
10 kDa
40 kDa 20 kDa
60 kDa 15 kDa
70% 20 kDa
15 kDa
10 kDa 50 kDa
15 kDa
80% 15 kDa
50 kDa
10 kDa
20 kDa 20 kDa
10 kDa 15 kDa
166 kDa 66 kDa 30 kDa 30 kDa
90% 10 kDa
15 kDa
40 kDa
20 kDa
55 kDa
15 kDa
97 kDa 10 kDa
100%
Figure 11. Migration patterns of protein molecular weight standards in Novex Tris-glycine gels. For optimal results, protein bands should
migrate within the gray shaded areas. (A) *Migration patterns of HiMark™ Unstained Protein Standard. † Migration patterns of Novex Sharp Pre-
Stained Protein Standard or Novex Sharp Unstained Protein Standard. (B) † † Migration pattern of NativeMARK Unstained Protein Standard.
For sample cleanup prior to electrophoresis, we recommend using the Buffers for native proteins: Invitrogen™ Novex™ Tris-Glycine Native
Pierce SDS-PAGE Sample Prep Kit. Sample Buffer and Novex™ Tris-Glycine Native Running Buffer.
Buffers for denatured proteins: Invitrogen™ Novex™ Tris-Glycine SDS PageRuler, PageRuler Plus, and Spectra protein ladders
Sample Buffer and Novex™ Tris-Glycine SDS Running Buffer. are recommended for molecular weight determination with
Novex Tris-Glycine gels.

NativePAGE gels
Superior resolution of native proteins and
protein complexes
The Invitrogen™ NativePAGE™ Bis-Tris gel system

is based on the blue native polyacrylamide gel Specifications
electrophoresis (BN PAGE) technique that uses
• Shelf life: 6 months
Coomassie G-250 dye as a charge shift molecule
that binds to proteins and confers a negative • Average run time: 90 minutes
charge without denaturing the proteins (Figure • Separation range: 15–10,000 kDa
12). This technique overcomes the limitations
• Polyacrylamide concentrations: gradient 3–12% and 4–16%
of traditional native electrophoresis by providing
a near-neutral operating pH and detergent • Gel dimensions: 8 x 8 cm (1 mm thick)
compatibility. The near-neutral (pH 7.5) environment • Maximum sample volume per 10-well gel: 25 μL
of the NativePAGE system during electrophoresis
results in maximum protein and gel matrix stability,
enabling better band resolution than other native
Figure 12. NativePAGE gel
gel systems. A gel migration chart is shown in electrophoresis. Two-fold
Figure 13. Is there a higher res pic dilution series of protein extracts
somewhere? I copied and were run on an Invitrogen™
pasted this from source file. NativePAGE™ Novex™ 3–12% Bis-
The NativePAGE gel system is designed for: Tris Protein Gel using a Mini Gel
Tank. Following electrophoresis,
• A wide resolving range—from 15 kDa to over 10 MDa
the gel was stained with
(Figure 12), regardless of isoelectric point Coomassie dye and imaged
using a flatbed scanner. Lanes 1
• Neutral-pH separation—the native state of protein com- and 10: blank; Lanes 2 and 6:
plexes is better preserved 5 μL NativeMark Unstained
ts acquired w
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• Superior performance—higher resolution than Tris-glycine and 5: 10, 5, and 2.5 μg spinach
native electrophoresis chloroplast extract; Lanes 7, 8
and 9: 10, 5, and 2.5 μg bovine

th e
Mini Gel Tan
k mitochondrial extract.
Advantages of the NativePAGE gel system over
the Tris-glycine gel system include:
• Reduced vertical streaking—Coomassie G-250 dye binds
to nonionic detergent molecules in the sample and carries
them in the dye front, ahead of resolving proteins
• Better separation of proteins—positively charged pro-

teins with high isoelectric points are converted to proteins
with a net negative charge, allowing migration to the anode
• Minimized protein aggregation—Coomassie G-250 dye

binding allows separation of membrane proteins and
proteins with exposed hydrophobic areas
Learn more at thermofisher.com/nativepage
NativePAGE gel
Did you know?

The blue native polyacrylamide
gel electrophoresis technique
was developed by Hermann
Schagger and Gebhard von
Jagow in 1991.
Figure 13. NativePAGE gel migration chart.

Migration patterns of the NativeMark Unstained
Protein Standard on NativePAGE gels
are shown.
NativeMark Unstained Protein Standard is recommended for use
with native gel chemistries, including our Tris-glycine, Tris-acetate, and
NativePAGE gel systems. This standard offers a wide molecular weight
range of 20–1,200 kDa, and the 242 kDa β-phycoerythrin band is visible as
a red band after electrophoresis for reference (prior to staining). See page
40 for details.

Novex Tricine gels

High-resolution gels for peptide analysis
and low molecular weight proteins
The Invitrogen™ Novex™ Tricine gel system is a Specifications

modification of the Tris-glycine system in which • Shelf life: 1–2 months
tricine replaces glycine in the running buffer.
• Average run time: 90 minutes
This system uses a discontinuous buffer system
specifically designed for the resolution of low • Separation range: 2–20 kDa
molecular weight proteins (Figure 14). • Polyacrylamide concentrations: fixed 10% and 16%;
gradient 10–20%
Advantages of Novex Tricine gels over
Tris-glycine gels include:
• Maximum sample volume per 10-well gel: 25 μL
• Increased resolution of proteins with molecular weights
as low as 2 kDa (Figure 15)
ts acquired w
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• Improved compatibility with direct protein sequencing Re

applications after transferring to PVDF membranes
• Minimized protein modification due to the lower pH of

th e k
the tricine buffering system Mini Gel Tan
Figure 14. Novex Tricine

Good to know
standards and samples
How Novex Tricine gels work were loaded at 10 μL sample
volumes on Invitrogen™ Novex™
10–20% Tricine Protein Gels.
In the traditional Tris-glycine protein gel system, the resolution Electrophoresis was performed
of smaller proteins (<10 kDa) is hindered by the continuous using the Mini Gel Tank at 200 V (constant). Sharp, straight bands were
accumulation of free dodecyl sulfate (DS) ions from the SDS observed after staining with SimplyBlue SafeStain. Images were acquired
using a flatbed scanner. Lane 1: SeeBlue Plus2 Prestained Standard; Lane
sample and running buffers in the stacking gel, which causes 2: 10 μg E. coli lysate; Lane 3: Mark12 Unstained Standard (blend of 12
mixing of the DS ions with smaller proteins and results in fuzzy purified proteins); Lane 4: 40 μg HeLa cell lysate; Lane 5: 20 μg HeLa cell
bands and decreased resolution. The mixing also interferes lysate; Lane 6: 5 μg BSA; Lane 7: 40 μg Jurkat cell lysate; Lane 8: 5 μg
GST fusion protein; Lane 9: Novex Sharp Unstained Protein Standard; Lane
with the fixing and staining of smaller proteins. The Novex 10: 5 μg β-galactosidase.
Tricine gel system uses a low pH in the gel buffer and sub-
stitutes tricine for glycine in the running buffer. The smaller
proteins and peptides that migrate with the stacked DS ions
in the Tris-glycine gel system are well separated from DS ions
in the Novex Tricine gel system, offering sharper bands and
higher resolution.
Learn more at thermofisher.com/tricine
Novex Tricine gel
Did you know?

Sample preparation is not
the only factor that can result
in poorly resolved bands.
You can minimize protein
degradation by using gels
with neutral-pH chemistry.
Figure 15. Novex Tricine gel migration chart.
For optimal resolution, protein bands should
migrate within the shaded areas.
Use Novex Tricine gels with our In-Gel Tryptic Digestion Kit for separation
and digestion of peptides for mass spectrometry.

Novex IEF gels

Precast gels for isoelectric
point determination
Isoelectric focusing (IEF) is an electrophoresis Specifications

technique that separates proteins based on their • Shelf life: 2 months
isoelectric point (pI). The pI is the pH at which a
• Average run time: 2.5 hours
protein has no net charge and does not move in an
electric field. Invitrogen™ Novex™ IEF gels effectively • Separation range:
create a pH gradient so proteins separate —pH 3–10 gels: pI performance range is 3.5–8
according to their unique pI (Figure 16 and 17). —pH 3–7 gels: pI performance range is 3.0–7.0
These gels can be used for pI determination or • Polyacrylamide concentration: fixed 5%
for detection of minor changes in a protein due to
deamination, phosphorylation, or glycosylation, and
can resolve different proteins of similar size that • Maximum sample volume per 10-well gel: 20 μL
cannot be resolved on standard SDS-PAGE gels.
ts acquired w
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When used with our convenient, pre-optimized

1 2 3 4 5 6 7 8 9 10
buffers, solubilizers, and molecular weight pl
markers, Novex IEF gels can provide: 8.0

th e k
Mini Gel Tan
• Accurate pI determination
• Clear, sharp bands for easy identification of 7.4

protein modifications Figure 16. Novex IEF
gel electrophoresis.
• Higher resolution of slight differences in size when used in 6.0
A 2-fold dilution series
of IEF Marker 3–10
combination with SDS-PAGE for 2D electrophoresis
was run in duplicate
4.5 on an Invitrogen™
Novex™ pH 3–10 IEF
Protein Gel using
a Mini Gel Tank. The IEF Marker 3–10 consists of proteins with a variety
of isoelectric points; these proteins include lectin (pI = 7.8, 8.0, and 8.3),
myoglobin from horse muscle (pI = 6.9 and 7.4), carbonic anhydrase from
bovine erythrocytes (pI = 6.0), β-lactoglobulin from bovine milk (pI = 5.2 and
5.3), soybean trypsin inhibitor (pI = 4.5), and glucose oxidase (pI = 4.2). After
electrophoresis, the gel was fixed and stained using Coomassie R-250 dye.
Gel imaging was performed with a flatbed scanner. Volume of IEF Marker
3–10 loaded: Lanes 1 and 6: 20 μL; Lanes 2 and 7: 10 μL; Lanes 3 and
8: 5 μL; Lanes 4 and 9: 2.5 μL; Lanes 5 and 10: blank.
Learn more at thermofisher.com/ief
Novex IEF gel
Separated on
precast vertical
gel (slab)
Cathode –
Did you know?

Harry Svensson-Rilbe and
his student Olof Vesterberg
first described the theory
of separation of amphoteric
proteins along a pH gradient
by applying an electric field
in the 1960s.
Anode +
Figure 17. Novex IEF gel migration chart using

the Novex IEF marker. Proteins shown are
1: amyloglucosidase (Aspergillus niger), pI = 3.5;
2: glucose oxidase (Aspergillus niger), pI = 4.2;
3: trypsin inhibitor (soybean), pI = 4.5; 4a and 4c:
β-lactoglobulin (bovine, milk), pI = 5.2 and 5.3;
5: carbonic anhydrase (bovine, erythrocytes), pI
= 6.0; 6a and 6c: myoglobin (horse, muscle), pI =
6.9 and 7.4; 7a, 7m and 7c: lectin (Lens culinaris),
pI = 7.8, 8.0 and 8.3; 8: ribonuclease A (bovine,
pancreas), pI = 9.5; and 9: cytochrome c (horse,
heart), pI = 10.7.
Novex IEF buffer kits—includes optimized cathode, anode, and sample ZOOM™ IEF Fractionator Combo Kit—
buffers to reduce variability and enable consistent results. offers a fast, reliable method to reduce
sample complexity, enrich low-abundance
IEF Marker 3–10—ready to use, enables accurate results. proteins, and increase the dynamic range
of detection.

Novex Zymogram gels Table 1. Novex Zymogram gels available.
Novex
Novex Novex
Zymogram
Easy in-gel protease analysis Zymogram Zymogram
blue casein
gelatin gel casein gel
gel
Invitrogen™ Novex™ Zymogram gels are excellent
Gel 10% Tris- 12% Tris- 4–16% Tris-
tools for detecting and characterizing proteases composition Glycine gel Glycine gel Glycine gel
that utilize casein or gelatin as a substrate.
Casein and gelatin are the most commonly Substrate 0.1% gelatin 0.05% casein 0.1% casein,
used substrates for demonstrating the activity with blue stain
incorporated
of proteases. Novex Zymogram gels are used
in gel
to analyze a variety of enzymes, including matrix
metalloproteinases, lipases, and other proteases
(Figure 18). Available gel types are shown in Table 1. Sensitivity 10–6 units of 7 x 10 –4 units 1.5 x 10 –3 units
collagenase of trypsin of trypsin
Good to know
Post-staining Yes Yes No

How do Novex Zymogram gels work?
required?
Protease samples are denatured in SDS buffer under nonre- Separation 20–120 kDa 30–150 kDa 10–220 kDa
ducing conditions and without heating, and run on a Novex range
Zymogram gel using Novex Tris-Glycine SDS Running Buffer.
After electrophoresis, the enzyme is renatured by incubating
the gel in Invitrogen™ Novex™ Zymogram™ Renaturing Buffer Specifications
that contains a nonionic detergent. The gels are then equili- • Shelf life: 2 months
brated in Invitrogen™ Novex™ Zymogram™ Developing Buffer
• Average run time: 90 minutes
to add divalent metal cations required for enzymatic activity,
and then stained and destained. Regions of protease activity • Separation range: 10–220 kDa (Figure 19)
appear as clear bands against a dark blue background where • Polyacrylamide concentrations: fixed 10% (with gelatin), fixed
the protease has digested the substrate. 12% (with casein); gradient 4–16% (with blue casein)
• Maximum sample volume per well: 20 μL
ts acquired w
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1 2 3 4 5 6 7 8 9 10 Figure 18. Novex Zymogram gel electrophoresis.

Type I collagenase was run in duplicate on an
T
Invitrogen™ Novex™ 10% Zymogram (Gelatin) Protein

th e
Mini Gel Tan
k Gel using a Mini Gel Tank. The gel was developed
using Novex Zymogram Renaturing Buffer and Novex
Zymogram Developing Buffer and stained using SimplyBlue
SafeStain. Images were acquired using a flatbed scanner. Lanes 3 and 7:
5 μL of 2.0 μU/mL type I collagenase; Lanes 1, 4, 5, and 10: 12 μL SeeBlue
Prestained Protein Standard.
Learn more at thermofisher.com/zymogram
Novex Zymogram gel
Protease analysis
4—16% gel
10% gel 12% gel
(w/prestained
(w/gelatin) (w/casein)
casein blue)
10
20
30
40
% of length of gel
50
2 1 Figure 19. Novex Zymogram gel

migration chart. The numbered bands
60 refer to the following proteases:
2 2 Band 1: Collagenase Type I (140 kDa)
Band 2: Thermolysin (37 kDa)
3 3 Band 3: Chymotrypsin (30 kDa)
70 Band 4: Trypsin (19 kDa)
4
3
4
2
80
4
90
4
100
After electrophoresis, incubate the gel in Zymogram Renaturing
Buffer to renature the enzyme. The gels are then equilibrated in
Zymogram Developing Buffer to add divalent metal cations required
for enzymatic activity.

E-PAGE High-Throughput
Precast Gel System
Protein separation and analysis for
increased sample throughput
The Invitrogen™ E-PAGE™ High-Throughput

Precast Gel System is designed for fast, bufferless
medium- and high-throughput protein analysis.
Invitrogen™ E-PAGE™ 48-well and 96-well precast
gels consist of a buffered gel matrix and electrodes
packaged inside a disposable, UV-transparent Specifications
cassette. Each cassette is labeled with a unique
barcode to facilitate identification of the gel using • Shelf life: 6 months
commercial barcode readers. These gels can be • Average run time: 14 minutes
loaded by multichannel pipettor or automated • Separation range: 10–200 kDa
loading system. The E-PAGE system also includes
E-Base™ integrated devices to run the gels, an • Polyacrylamide concentrations:
– E-PAGE™ 48 gel: fixed 8%
E-Holder™ platform for optional robotic loading, and
– E-PAGE™ 96 gel: fixed 6%
free E-Editor™ 2.0 Software to align images for easy
comparison. • Gel dimensions: 13.5 x 10.8 cm (3.7 mm thick)
• Maximum sample volume per well:

Advantages of using the E-PAGE High-Throughput – E-PAGE 48 gel: 20 μL
Precast Gel System include: – E-PAGE 96 gel: 15 μL
• Ease-of-use—quick setup and fast protein separation in
A B
about 23 minutes
• Fast loading—compatible with multichannel pipettors and

robotic loading
• Efficient western blotting and staining—optimized

protocols and reagents
Good to know C
Mother E-Base E-PAGE 96 gels

How do E-PAGE gels work?
E-PAGE gels run in the Invitrogen™ E-Base electrophoresis de- Daughter E-Base
vice, which has an integrated power supply for direct connec-

tion to an electrical outlet. Use the Invitrogen™ Mother E-Base™
Figure 20. Loading and running E-PAGE gels. (A) Loading E-PAGE 48
device for a single E-PAGE gel, or use the Mother E-Base gels using a multi-channel pipettor. (B) Loading E-PAGE 96 gels using a
device in conjunction with two or more Invitrogen™ Daughter multi-channel pipettor. (C) The Mother/Daughter E-Base combination.
E-Base™ devices for running multiple gels simultaneously.
Learn more at thermofisher.com/epage
For ordering information refer to page 85.

E-PAGE gel
E-PAGE E-PAGE
48 8% Gel 96 6% Gel
0% 0%
220 kDa
10%
25%
120 kDa
220 kDa
Did you know?
20% Our E-Base devices are
60 kDa
120 kDa 50% compatible with the Society for
40 kDa
30%
100 kDa Biomolecules Screening (SBS)
standard plate size and can be
80 kDa conveniently mounted on liquid
75%
40% 20 kDa Figure 21. E-PAGE gel migration chart.
handling robot decks.
Migration patterns of the Invitrogen™ E-PAGE™
60 kDa
MagicMark™ Unstained Protein Standard
50 kDa
50% 100% are shown.
40 kDa
60%
30 kDa
70%
20 kDa
80%
90%
100%
The E-PAGE™ SeeBlue™ Prestained Protein Standard or E-PAGE
MagicMark Unstained Protein Standard are specifically designed for use
with E-PAGE gels.

Prepare the sample

Sample prep kits High salt concentration in samples: High salt concentrations
result in increased conductivity that affects protein migration, and
can result in gel artifacts in adjacent lanes containing samples with
Before a sample can be loaded normal salt concentrations. Perform dialysis or precipitate and
onto a gel for analysis, it must resuspend samples in lower-salt buffer prior to electrophoresis.
be properly prepared. Depending Guanidine-HCl in samples: Samples solubilized in guanidine-

HCl have high ionic strength, and produce increased conductivity
on the gel type, this may involve similar to high salt concentrations. In addition, guanidine
precipitates in the presence of SDS leading to various types of gel
denaturing the proteins, reducing artifacts. If possible, change the solubilization agent by dialysis
prior to electrophoresis.
any disulfide bonds, adjusting
the ionic strength, and removing Cell lysates
Consider the following when performing electrophoresis of
interfering contaminants. General cell lysates:
guidelines for preparing samples • Genomic DNA in the cell lysate may cause the sample to
become viscous and affect protein migration patterns and
are provided below. resolution. Shear genomic DNA to reduce viscosity before
loading the sample.
• Cells lysates contain soluble and insoluble fractions. The size of

General guidelines for each fraction depends on the type of sample being analyzed.
preparing samples: The nature of the insoluble fraction may result in altered
protein migration patterns and resolution. Separate the two
fractions by centrifugation and load them on separate lanes for
Prepare your sample in the appropriate sample buffer such that
electrophoresis.
the final concentration of the sample buffer is 1X. Recommended
sample buffers are listed on page 29. • If radioimmunoprecipitation assay (RIPA) buffer is used in cell
lysis, subsequent blotting of proteins less than 40 kDa may be
inhibited due to the presence of Triton™ X-100 in the buffer.
Running reduced and non-reduced samples: For optimal
results, we do not recommend running reduced and non-reduced For quick protein clean-up and enrichment for SDS-PAGE we
samples on the same gel. If you do choose to run reduced and recommend using the Thermo Scientific Pierce SDS-PAGE Sample
non-reduced samples on the same gel, do not run reduced and Prep Kit, which removes substances such as guanidine-HCL
non-reduced samples in adjacent lanes. The reducing agent may and ionic detergents that can result in protein bands that appear
have a carry-over effect on the non-reduced samples if they are in smeared or wavy in the gel or on a western blot.
close proximity.
Heating samples: Heating the sample at 100°C in SDS-containing

buffer results in proteolysis (Kubo, 1995). We recommend heating
samples for denaturing electrophoresis (reduced or non-reduced)
at 85°C for 2–5 minutes for optimal results. Do not heat the
samples for non-denaturing (native) electrophoresis or Novex
Zymogram Gels.

Pierce SDS-PAGE M S
Untreated
M S
Sample treated with the Pierce
SDS-PAGE Sample Prep Kit
M S M S
Sample Prep Kit

Quick protein clean-up and enrichment
for SDS-PAGE
A protein sample can be purged of any contaminants

typically in only 10 minutes using the Thermo Scientific™
Figure 22. Minimize distortion caused by detergents. Rat C6 cells were
Pierce™ SDS-PAGE Sample Prep Kit. This is much lysed and a membrane protein fraction isolated using the Thermo Scientific™
faster than dialysis or ultrafiltration and yields higher MemPER™ Eukaryotic Membrane Protein Extraction Reagent. Membrane
and hydrophilic cell fractions were separated by SDS-PAGE using 4–20%
protein recoveries while concentrating the sample.
gradient gels with or without prior treatment using the Pierce SDS-PAGE
Sample Prep Kit. Western blot analysis was performed using an antibody
Advantages of using the Pierce SDS-PAGE Sample against cytochrome oxidase subunit 4 (COX4) and Thermo Scientific™
SuperSignal™ West Femto chemiluminescent substrate. Kit-treated samples
Prep Kit include:
exhibit better band straightness and resolution with low molecular weight
• Eliminates artifacts caused by incompatible proteins than samples that were untreated.
S = Soluble fraction (hydrophilic) M = Membrane fraction
contaminants—removes dyes, reducing agents, deter-
gents, sugars, glycerol, guanidine, urea, and ammonium 100 Figure 23.
sulfate to provide reproducible results for SDS-PAGE Consistent
Percent protein recovered
80 88% protein
analysis (Figure 22) 85%
75% 77% 77% recovery is
74%
• Compatible with the Thermo Scientific™ Pierce™ BCA 60 achieved
Assay—allows quantification of the processed sample using the
40 Pierce SDS-
• Enriches dilute protein solutions—concentrates protein PAGE Sample
20 Prep Kit.
sample by eight-fold in less than 20 minutes for SDS-PAGE
Pure proteins
analysis (Figure 22) (60 μg) of
0
Carbonic Ovalbumin Transferrin Ubiquitin Cytochrome c Bacterial assorted
• Fast and easy to use for up to 70 μg of protein per anhydrase lysat e molecular mass
sample—uses new spin cup format that allows higher (30, 44, 80, 86, and 120 kDa) as well as a bacterial lysate were processed
amounts of protein to be processed than with the original using this kit. Protein concentrations were determined with the Pierce BCA
procedure Protein Assay Kit and reported as percent protein recovered.
Table 2. Interfering substances effectively removed.

Good does
How to know
it work?
Percent protein recovered
Interfering reagents (Starting amount = 20 μg BSA)
Our Pierce SDS-PAGE Sample Prep Kit uses a unique resin
Control (water) 75%
of modified diatomaceous earth that binds protein in DMSO. 0.5 M Sodium chloride 80%
Simply combine 2–300 μL of sample containing up to 70 μg of 2 M Ammonium sulfate 76%
protein with 20 μL of Pierce™ SDS Protein Binding Resin and 20% SDS 75%
DMSO. After the proteins bind to the resin, wash away the 10% Triton™ detergent 75%
unbound contaminating chemicals. Finally, elute the sample 6 M Urea:DMSO (1:3 ratio) 75%
1 M Sodium chloride 75%
in 50 μL of the Elution Buffer. The recovered protein sample is
6 M Urea 74%
ready to mix with the supplied Sample Loading Buffer for
10% CHAPS 80%
gel loading. 25% Glycerol 71%
10% OTG 71%
Learn more at 2 M Guanidinium•HCl 70%

40% Sucrose 70%
thermofisher.com/PAGEsampleprep
Select buffers
Buffers and reagents
Protein samples prepared for If suitable, negatively charged,
PAGE analysis are denatured low molecular weight dye is also
by heating in the presence of included in the sample buffer;
a sample buffer with or without it will migrate at the buffer front,
a reducing agent. The protein enabling one to monitor the
sample is mixed with the sample progress of electrophoresis.
buffer and heated for 2–10 The most common tracking dye
minutes, then cooled to room for sample loading buffers is
temperature before it is applied bromophenol blue.
to the sample well on the gel.
We offer premixed, reliable
Loading buffers also contain
SDS-PAGE buffers and reagents
glycerol so that they are heavier
including sample buffers,
than water and sink neatly to the
running buffers, reducing agents,
bottom of the buffer-submerged
and antioxidants.
well when added to a gel.
Learn more at
thermofisher.com/electrophoresisbuffers

Recommended SDS-PAGE buffers and reagents

Sample buffer Other Running buffer
optimized for use compatible optimized for use
Gel type with the gel sample buffers with the gel
Bolt Bis-Tris • Bolt™ Sample Reducing • Pierce™ LDS • Bolt™ MES SDS MES vs. MOPS Running Buffer:
Plus Gel Agent (10X) Sample Buffer (4X) Running Buffer (20X) • Use MES SDS running buffers
• Bolt™ LDS Sample Buffer for storage at RT • Bolt™ MOPS SDS Running to resolve small molecular
(4X) (nonreducing) Buffer (20X) weight proteins.
• Bolt Antioxidant • Pierce™ Lane • Use MOPS running buffers to resolve
Marker Non- mid-size proteins.
™
NuPAGE • NuPAGE Sample Reducing Sample • NuPAGE™ MES SDS
Bis-Tris Gel Reducing Agent (10X) Buffer (5X)— Running Buffer (20X) MES has a lower pKa than MOPS,
• NuPAGE Antioxidant storage at RT; • NuPAGE™ MOPS SDS enabling gels with MES running buffer
• NuPAGE™ LDS Sample when you desire to Running Buffer (20X) to run faster than gels with MOPS SDS
Buffer (4X) (nonreducing) dilute your sample running buffer. The difference in ion
less and require migration affects stacking and results in
transferable marker a difference in protein separation range
dye to nitrocellulose between these buffers.
membranes
NuPAGE • Novex Tris-Glycine SDS • NuPAGE™ Tris-Acetate SDS Reducing agent:

• Pierce™ Lane
Tris-Acetate Gel Sample Buffer (2X) Running Buffer (20X) When preparing samples for
Marker Reducing
• NuPAGE Sample • Novex Tris-Glycine Native reducing gel electrophoresis, any of the
Sample Buffer
Reducing Agent (10X) Running Buffer (10X) following reducing agents may
(5X)—when you
• Novex Tris-Glycine Native be used:
desire to dilute your
Sample Buffer (2X) • Bolt Sample Reducing Agent
sample less and
Novex Tris-Glycine • Novex Tris-Glycine SDS require transferable • Novex Tris-Glycine SDS • NuPAGE Sample Reducing Agent
Gel Sample Buffer (2X) marker dye to Running Buffer (10X)
nitrocellulose Dithiothreitol (DTT), 50 mM final
• NuPAGE Sample • Novex Tris-Glycine Native
membranes concentration
Reducing Agent Running Buffer (10X)
• Novex Tris-Glycine Native • Pierce™ Tris-Glycine SDS • β-mercaptoethanol (β-ME), 2.5%
Sample Buffer (2X) Buffer (10X) final concentration
• BupH™ Tris-Glycine-SDS
• tris(2-carboxyethyl)phosphine (TCEP),
Buffer Packs
50 mM final concentration
Novex Tricine Gel • Novex™ Tricine SDS Sample • Novex Tricine SDS Running
Add the reducing agent to the sample
Buffer (2X) Buffer (10X)
up to an hour before loading the gel.
Avoid storing reduced samples for long
periods, even if they are frozen.
NativePAGE Gel • NativePAGE™ Sample Buffer • NativePAGE™ Running
Reoxidation of samples can occur
(4X) Buffer (20X)
during storage and produce
• NativePAGE™ 5% G-250 • NativePAGE™ Cathode
inconsistent results.
Sample Additive Buffer Additive (20X)
Novex IEF Gel • Novex™ IEF Sample Buffer, • Novex™ IEF Anode Buffer
pH 3–10 (2X) (50X)
• Novex™ IEF Sample Buffer, • Novex™ IEF Cathode Buffer,
pH 3–7 (2X) pH 3–10 (10X)
• Novex™ IEF Cathode Buffer,
pH 3–7 (10X)
Novex Zymogram • Novex Tris-Glycine SDS • Novex Tris-Glycine SDS

Gels* Sample Buffer (2X) Running Buffer (10X)
*Novex Zymogram Developing Buffer (10X) and Novex Zymogram Renaturing Buffer (10X) are available for
visualizing the Novex Zymogram gels.

Buffer recipes
NuPAGE buffer recipes
Buffer Storage Component Concentration (1X)
NuPAGE LDS Sample Buffer +4˚–25˚C
Glycerol 0%
Tris base 141 mM
Tris HCl 106 mM
LDS 2%
EDTA 0.51 mM
SERVA™ Blue G-250 0.22 mM
Phenol Red 0.175 mM
(pH 8.5)
NuPAGE MOPS SDS Running Buffer* +4˚–25˚C

MOPS 50 mM
Tris base 50 mM
SDS 0.1%
EDTA 1 mM
(pH 7.7)
NuPAGE MES SDS Running Buffer* +4˚–25˚C

MES 50 mM
Tris base 50 mM
SDS 0.1%
EDTA 1 mM
(pH 7.3)
NuPAGE™ Transfer Buffer +4˚–25˚C

Bicine 25 mM
Bis-Tris (free base) 25 mM
EDTA 1.0 mM
Chlorobutanol 0.05 mM
(pH 7.2)
NuPAGE Tris-Acetate SDS Running Buffer +4˚–25˚C

Tris base 50 mM
Tricine 50 mM
SDS 0.1%
(pH 8.24)
* The pre-mixed buffers (Cat. Nos. NP0001 and NP0002) also contain trace amounts of the proprietary NuPAGE Antioxidant (Cat. No. NP0005) for
stability. Additional Antioxidant may be required with specific protocols.

Tris-glycine buffer recipes

Tris-Glycine SDS Sample Buffer +4˚C
Tris HCl* 63 mM
Glycerol 10%
SDS 2%
Bromophenol Blue 0.0025%
Deionized water —
(pH 6.8)
Tris-Glycine Native Sample Buffer +4˚C

Tris HCl* 100 mM
Glycerol 10%
Deionized water —
(pH 8.6)
Tris-Glycine SDS Running Buffer Room

temperature Tris base 25 mM
Glycine 192 mM
SDS 0.1%
Deionized water —
(pH 8.3)
Tris-Glycine Native Running Buffer Room

Glycine 192 mM
Deionized water —
(pH 8.3)
Tris-Glycine Transfer Buffer Room

Glycine 96 mM
Deionized water —
(pH 8.3)
* Tris HCl solutions are prepared from Tris base and pH adjusted with 6 N HCl.

Buffer recipes
Tricine buffer recipes
Tricine SDS Sample Buffer +4˚C
Tris HCl* 450 mM
Glycerol 12%
SDS 4%
Coomassie Blue G 0.0075%
Phenol Red 0.0025%
Deionized water –
(pH 8.45)
Tricine SDS Running Buffer Room

Tricine 100 mM
SDS 0.1%
Deionized water –
(pH 8.3)
Zymogram buffer recipes

Zymogram Room
Renaturing Buffer temperature Triton™ X-100 2.7% (w/v) in H2O
Deionized water
Zymogram Room
Developing Buffer temperature Tris HCI* 50 mM
NaCl 200 mM
CaCl2•2 H2O 5 mM
Brij™ 35 0.006% (w/v)
Deionized water _
(pH 7.6)

Isoelectric focusing buffer recipes

IEF Sample Buffer pH 3-7 +4˚C
Lysine (free base) 40 mM
Glycerol 15%
Deionized water —
IEF Sample Buffer pH 3-10 +4˚C

Arginine (free base) 20 mM
Glycerol 15%
Deionized water —
IEF Cathode Buffer pH 3-7 +4˚C

(upper buffer chamber) Lysine (free base) 40 mM
Deionized water —
IEF Cathode Buffer pH 3-10 +4˚C

(upper buffer chamber) Arginine (free base) 20 mM
Deionized water —
(pH 10.1)
IEF Anode Buffer (for both pH ranges) Room

(lower buffer chamber) temperature Phosphoric acid 85% 7 mM
Deionized water —
(pH 2.4)
Urea-Thiourea-CHAPS –20˚C
(rehydration buffer for IPG strips) Deionized urea 7M
Deionized thiourea 2M
CHAPS 2–4%
Ampholytes* 0.2–2.0%
Ultrapure water —
DTT 20 mM
* For ZOOM™ Strip pH 9-12 use 1% ZOOM™ Focusing Buffer pH 7-12 instead of ampholytes.

Select the standard

Protein ladders and standards
To assess the relative molecular each marker protein migrates
weights (sizes) of proteins in a through the gel. After measuring
sample, a mixture containing the migration distance that an
several proteins of known unknown protein travels through
molecular mass are run alongside the same gel, its molecular weight
the test sample lane(s). Often can be determined graphically from
these protein mixtures are run the standard curve.
on the outer lanes of the gel, to
Several kinds of ready-to-use
maximize the number of remaining
protein molecular weight (MW)
gel wells for test samples, but can
markers are available that are
also be useful in the middle wells
labeled, prestained, or unstained
of the gel when running a large
for different modes of detection
gel with many wells. Such sets of
and downstream applications. We
known protein mixtures are called
offer ladders suitable for both SDS-
protein molecular weight markers
PAGE as well as native PAGE.
or protein ladders. It is important
to choose a protein ladder that
consists of proteins with molecular
weights that span the molecular
weight range of the protein(s) of
interest. A standard curve can be
constructed from the distances
Ready-to-use prestained and unstained protein

Unstained protein ladders
ladders with exceptional lot-to-lot consistency
Low range PageRuler Unstained Low Range Protein Ladder
Broad range PageRuler Unstained Protein Ladder We offer a broad range of prestained and unstained protein
High range NativeMark Unstained Protein Standard ladders supplied in a ready-to-use format to facilitate easy protein
analysis during gel electrophoresis and western blotting (Table 3).
Recommended for:
• Precise determination of target protein molecular weight All of our protein ladders offer:
• Performance—sharp protein bands and consistent migration

patterns provide easy molecular weight determination
Prestained protein ladders • Convenience—protein ladders are ready to load, with no
Low range PageRuler Prestained Protein Ladder heating required
Broad range PageRuler Plus Prestained Protein Ladder • Reliability—exceptional lot-to-lot consistency
Spectra™ Multicolor Broad Range Protein Ladder and reproducibility
High range HiMark Prestained Protein Standard
Spectra Multicolor High Range Protein Ladder
Recommended for: Learn more at
• Approximate determination of molecular weight thermofisher.com/proteinstandards
• Monitoring the progress of electrophoresis runs
• Estimating the efficiency of protein transfer to the membrane during
western blotting
Other
Western MagicMark XP Western Protein Standard
Specialty PageRuler Prestained NIR Protein Ladder

BenchMark Fluorescent Protein Standard
BenchMark His-tagged Protein Standard
IEF Marker 3-10

Table 3. Protein standard selection guide

No. of Protein MW Protein band
Category Product Range bands Reference bands determination visualization
Unstained ladders and standards
Unstained standards PageRuler Unstained Low 3.4–100 kDa 8 25 kDa Best NA
Range Protein Ladder
PageRuler Unstained Protein 10–200 kDa 14 50 kDa Good NA

Ladder
NativeMark Unstained Protein 20–1,200 kDa 8 Best for native NA

Standard electrophoresis
Pretained protein ladders

Prestained protein PageRuler Prestained Protein 10–180 kDa 10 Green 10 kDa; orange Good Good
standards Ladder 70 kDa
PageRuler Plus Prestained 10–250 kDa 9 Green 10 kDa; orange Good Good
Protein Ladder 25 and 70 kDa
HiMark Prestained Protein 30–460 kDa 9 Best for high MW Good

Standard proteins
Spectra Multicolor Broad Range 10–260 kDa 10 Green 10 and 50 kDa; Good Best
Protein Ladder orange 40, 70, and
260 kDa; pink 140 kDa
Spectra Multicolor High Range 40–300 kDa 8 Green 50 kDa; orange Good Best
Protein Ladder 70 and 300 kDa
Other ladders and standards

IEF IEF Marker 3-10 pI 3.5–10.7 13 Best for pI estimation NA
Chemiluminescent MagicMark XP Western Protein 20–220 kDa 9 Good NA

standard Standard
Near infrared (NIR) PageRuler Prestained NIR 11–250 kDa 10 55 kDa Good NA
standard Protein Ladder
Fluorescent BenchMark Fluorescent Protein 11–155 kDa 7 Good NA

standard Standard
His-tag standard BenchMark His-tagged Protein 10–160 kDa 10 Best NA

Standard
Learn more at
thermofisher.com/proteinstandards

Monitoring Coomassie dye, silver, Monitoring protein Chemiluminescent

electrophoresis run or fluorescent staining transfer band visualization
NA Best NA Good
NA Good NA Good
NA Best NA Good
Good NA Good Good
Good NA Good NA
Good NA Best for high MW proteins NA
Best NA Best NA
Best NA Best NA
NA Good NA NA
NA Good NA Best
NA NA NA NA
NA NA NA Good
NA Good NA Good for detection with

anti-His antibody

Unstained ladders and standards
PageRuler Unstained Low

Sharp bands and precise molecular
weight estimation for low molecular
weight proteins PageRuler Unstained Low Range Protein Ladder
NuPAGE 4–12% Bis-Tris Gel with MES SDS buffer
Thermo Scientific™ PageRuler™ Unstained Low

Range Protein Ladder is a mixture of eight proteins
and peptides for use as size standards that resolve
into clearly identifiable sharp bands when analyzed Storage specifications
by SDS-PAGE. The proteins (except for the 5 and • Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
3.4 kDa peptides) contain an integral Strep-tag™ II bromophenol blue, and glycerol
Sequence and may be detected on western blots
• Storage conditions: upon receipt store at –20°C
using Strep-Tactin™ Conjugates.
• Stability: 1 year from date of receipt
• Comprehensive—eight proteins and peptides spanning
3.4 to 100 kDa; the 25 kDa band is more intense than the
other bands for easy orientation
The PageRuler Unstained Protein Ladder is recommended for Novex
• Versatile—compatible with western blots by staining with Tris-Glycine, Bis-Tris or Tris-Acetate gels.
Ponceau S dye or Coomassie dye; compatible with Thermo
Scientific™ Pierce™ Reversible Protein Stain Kit for Nitrocellu-
lose Membranes or other protein stains
Learn more at
thermofisher.com/unstainedstandards

PageRuler Unstained
Protein Ladder
Sharp bands and precise molecular
weight estimation for a wide
range of proteins PageRuler Unstained Protein Ladder
Thermo Scientific™ PageRuler™ Unstained Protein

Ladder is a mixture of 14 recombinant, highly
purified, unstained proteins for use as size
standards in SDS-PAGE and western blotting. Each Storage specifications
protein in the ladder contains an integral Strep-tag • Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
II Sequence, which can be detected directly on bromophenol blue, and glycerol
western blots using a Strep-Tactin Conjugate or an
antibody against the Strep-tag II Sequence.
• Comprehensive—14 highly purified proteins with excellent
accuracy spanning 10 to 200 kDa; the ladder contains one
50 kDa reference band of higher intensity
The PageRuler Unstained Protein Ladder is recommended for Novex
• Versatile—compatible with Coomassie dye; compatible Tris-Glycine, Bis-Tris or Tris-Acetate gels.
with Pierce Reversible Protein Stain Kit for Nitrocellulose
Membranes, silver staining, or western blotting
Learn more at

NativeMark Unstained kDa kDa

1,236
Protein Standard
1,048
1,236
1,048 720
480
Convenient molecular weight estimation 720

242
for native electrophoresis 480

146 NativeMark Unstained
242 Protein Standard
66
NativePAGE Bis-Tris gels
146
™ ™
The Invitrogen NativeMark Unstained
66
Protein Standard is designed for molecular
20
weight estimation of proteins using native gel 20
electrophoresis. 3–12% 4–16%
• Comprehensive—contains a wide range of high molecular

Storage specifications
weight proteins, providing 8 protein bands in the range
of 20–1,200 kDa • Storage buffer: Bis/Tris-HCl (pH 7.0), NaCl, glycerol, and
Ponceau S
• Versatile—can be visualized using Coomassie, silver, or
fluorescent stains after electrophoresis, or with Ponceau S, • Storage conditions: upon receipt store at –20°C
Coomassie, or other membrane stains after western transfer • Stability: 6 months
The NativeMark Unstained Protein Standard is recommended for use
with NativePAGE Bis-Tris gels, Novex Tris-Glycine gels, or NuPAGE
Tris-Acetate gels.
Learn more at

Prestained ladders
PageRuler Prestained
Protein Ladder
Outstanding clarity for easy molecular
weight determination of low molecular
weight proteins PageRuler Prestained Protein Ladder
Thermo Scientific™ PageRuler™ Prestained Protein

Ladder is a mixture of 10 blue-, orange-, and
green-stained proteins for use as size standards
in SDS-PAGE and western blotting. The mobility Storage specifications
of prestained proteins can vary in different SDS- • Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
PAGE buffer systems; however, they are suitable for bromophenol blue, and glycerol
approximate molecular weight determination when
calibrated against unstained standards in the
same system. • Stability: 1 year from date of receipt
• Comprehensive—contains 10 proteins with a range of 10

to 180 kDa; includes one 70 kDa reference protein colored
with an orange dye and one 10 kDa reference protein The PageRuler Prestained Protein Ladder is recommended for use with
Tris-glycine, Bis-Tris, and Tris-acetate gels.
colored with a green dye
• Versatile—compatible with Coomassie dye staining and

western blotting
Learn more at
thermofisher.com/prestainedstandards

PageRuler Plus Prestained

Protein Ladder
Outstanding clarity for easy molecular
weight determination of a broad
PageRuler Plus Prestained Protein Ladder
range of proteins NuPAGE 4–12% Bis-Tris Gel with MES SDS buffer
Thermo Scientific™ PageRuler™ Plus Prestained

Protein Ladder is a mixture of 9 blue-, orange-, and
green-stained proteins for use as size standards in
SDS-PAGE and western blotting. The mobility of
prestained proteins can vary in different SDS-PAGE Storage specifications
buffer systems; however, they are suitable • Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
for approximate molecular weight determination bromophenol blue, and glycerol
when calibrated against unstained standards in
the same system.
• Comprehensive—9 proteins with a broad range of 10 to
250 kDa; includes 70 kDa and 25 kDa reference proteins
that are colored with an orange dye and one 10 kDa
reference protein that is colored with a green dye The PageRuler Plus Prestained Protein Ladder is recommended for Tris-
glycine, Bis-Tris, and Tris-acetate gels.
western blotting
Learn more at

HiMark Prestained kDa

460
Protein Standard 268

238
171
Superb analysis of high molecular 117
weight proteins
71
HiMark Prestained Protein Standard

The Invitrogen™ HiMark™ Prestained Protein 55
NuPAGE 3–8% Tris-acetate SDS buffer
Standard is designed for analysis of high molecular 41
weight proteins on NuPAGE Tris-acetate gels. 31
• Comprehensive—contains a wide range of high molecular

weight proteins, providing 9 protein bands in the range of
30–460 kDa
• Storage buffer: Tris-HCl, formamide, SDS, and phenol red
• Versatile—easy visualization of band migration during
electrophoresis and rapid evaluation of western • Storage conditions: upon receipt store at –20°C
transfer efficiency • Stability: 6 months from date of receipt
The HiMark Prestained Protein Standard is recommended for use with
NuPAGE Tris-Acetate gels under denaturing conditions. This standard
can also be used with NuPAGE 4–12% Bis-Tris gels with Invitrogen™
NuPAGE MOPS SDS Running Buffer and Novex 4% Tris-Glycine gels.
However, to obtain the best results with high molecular weight proteins,
always use NuPAGE Tris-Acetate gels.
The HiMark Prestained Protein Standard is also available as part of the

following kits that include gels, running and sample buffers, and stains or
blotting materials:
• Invitrogen™ NuPAGE™ Large Protein Staining Kit
• Invitrogen™ NuPAGE™ Large Protein Sensitive Staining Kit
• Invitrogen™ NuPAGE™ Large Protein Blotting Kit
Learn more at

Spectra Multicolor Broad

Superior visualization and analysis of a
broad range of proteins Spectra Multicolor Broad Range Protein Ladder
Thermo Scientific™ Spectra™ Multicolor Broad

Range Protein Ladder is a 4-color protein standard
containing 10 prestained proteins for use in gel
electrophoresis and western blotting. This standard
is designed for monitoring the progress of gels
during SDS-PAGE and for assessing western blot Storage specifications
transfer efficiency. Four different chromophores • Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
(blue, orange, green, and pink) are bound to the bromophenol blue, and glycerol
different component proteins, producing a brightly
colored ladder with an easy-to-remember pattern.
• Comprehensive—10 proteins with similar intensity span-
ning a broad range of 10 to 260 kDa
• Versatile—compatible with Coomassie dye staining and The Spectra Multicolor Broad Range Protein Ladder is
western blotting recommended for Tris-glycine, Bis-Tris, and
Tris-acetate gels.
Learn more at

Spectra Multicolor High

Superior and convenient visualization of
high molecular weight proteins
Spectra Multicolor High Range Protein Ladder
™ ™
Thermo Scientific Spectra Multicolor High Range
Protein Ladder is a mixture of 8 blue-, green-,
and orange-stained proteins for use as size
standards for high molecular weight proteins in gel
electrophoresis and western blotting. This marker Storage specifications
is designed for monitoring the progress of gels • Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
during SDS-PAGE, assessing western blot transfer bromophenol blue, and glycerol
efficiency, and estimating the approximate size of
proteins after gel staining or western blotting.
• Comprehensive—8 proteins of similar intensity spanning
a range of 40 to 300 kDa; 3 different chromophores (blue,
orange, and green) are bound to the different component
proteins, producing a brightly colored ladder with an The Spectra Multicolor High Range Protein Ladder is recommended for
Tris-glycine, Bis-Tris, and Tris-acetate gels.
easy-to-remember pattern

western blotting
Learn more at

Other ladders and standards
MagicMark XP Western kDa
220
Protein Standard 120

100
80
Accurate molecular weight estimation 60
50
directly on western blots 40

MagicMark XP Western Protein Standard
30
NuPAGE Bis-Tris gel, blotted to nitrocellulose,
and detected with Invitrogen™ WesternBreeze™
™
The MagicMark XP Western Protein Standard Chemiluminescent Kit
20
is specifically designed for easy and convenient
protein molecular weight estimation directly on
western blots. Each recombinant protein in the
standard contains an IgG binding site, which Storage specifications
binds the primary or secondary antibody used • Storage buffer: Tris-HCl (pH 6.8), DTT, glycerol, SDS, and
for detection of the target protein, allowing direct bromophenol blue
visualization of the standard on the western blot.
• Comprehensive—consists of 9 recombinant proteins from
• Stability: 4 months from date of receipt
20 to 220 kDa
• Versatile—compatible with chemiluminescent, Recommended products

chromogenic, and fluorescent detection The MagicMark XP Western Protein Standard is compatible
with a broad range of gels—NuPAGE Bis-Tris gels,
Novex Tris-Glycine gels, Novex Tricine gels, NuPAGE
Tris-Acetate gels, and Bolt Bis-Tris Plus gels.
Learn more at
thermofisher.com/westernblotstandard

PageRuler Prestained NIR

Protein Ladder
Sharp prestained standard for near-IR
fluorescent visualization and protein sizing
PageRuler Prestained NIR Protein Ladder
NuPAGE 4–12% Bis-Tris Gel with MES
Thermo Scientific™ PageRuler™ Prestained NIR SDS buffer
Protein Ladder is a mixture of 10 proteins that
are stained blue and labeled with a fluorophore Visual Infrared
for near-infrared (NIR) fluorescent visualization detection detection
and protein sizing following electrophoresis. The

molecular weight markers in this ladder resolve into Storage specifications
sharp bands when analyzed by SDS-PAGE. The • Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
55 kDa band is of greater intensity and serves as a bromophenol blue, and glycerol
reference band.
• Comprehensive—10 protein bands spanning 11 to
250 kDa
• Versatile—visualize using instruments equipped for Recommended products

detection of near-infrared fluorescence such as certain The PageRuler Prestained NIR Protein Ladder is recommended for visual
Typhoon™ Imagers and the LI-COR Odyssey™ Infrared detection, infrared imaging detection, and western blotting.
Imaging System; bands are directly visible because the
proteins are prestained blue
Learn more at
thermofisher.com/specialtystandards

BenchMark Fluorescent
Protein Standard
Efficient estimation of molecular weight by
fluorescent detection BenchMark Fluorescent Protein Standard
The Invitrogen™ BenchMark™ Fluorescent Protein

Standard consists of Alexa Fluor™ 488 dye–
conjugated proteins for molecular weight estimation
of fluorescently labeled proteins.
• Comprehensive—consists of 7 distinct protein bands in the
range of ~11–155 kDa • Storage buffer: Tris-HCl, SDS, glycerol, and Coomassie
Blue G-250
• Versatile—visualize on a UV transilluminator or laser-based
scanning instrument after SDS-PAGE • Storage conditions: upon receipt store at –20°C
• Stability: 6 months from date of receipt
The BenchMark Fluorescent Protein Standard is recommended for use
with NuPAGE gels or Novex Tris-Glycine gels.
Learn more at

BenchMark His-tagged
Protein Standard
Convenient detection and protein sizing
of His-tagged proteins
BenchMark His-tagged Protein Standard
NuPAGE 4–12% Bis-Tris Gel with MES
™ ™
The Invitrogen BenchMark His-tagged Protein SDS buffer
Standard can be used as a positive control and for
molecular weight sizing in His-tagged fusion protein SimplyBlue InVision
stain stain
detection. Each protein in the standard has a 6xHis
tag. Storage specifications
• Storage buffer: Tris-HCl, SDS, glycerol, DTT, and Coomassie
• Comprehensive—10 sharp and clear bands from 10 to 160
Blue G-250
kDa for molecular weight estimation of His-tagged proteins
• Versatile—can be visualized with Invitrogen™ InVision™ His-
Tag In-Gel Stain or Coomassie R-250 stain on SDS-PAGE • Stability: 6 months from date of receipt
gels, or with Anti-His (C-term) Antibody using chromogenic
or chemiluminescent detection systems Recommended products
The BenchMark His-tagged Protein Standard is recommended for use with
Learn more at NuPAGE gels and Novex Tris-Glycine gels.
IEF Marker 3-10

Accurate determination of protein
isoelectric points
The IEF Marker 3-10 is a ready-to-use protein IEF Marker 3-10

Novex pH 3–10 Gel
standard developed for IEF applications. This
marker can be used for monitoring of protein
separation on IEF gels and pI determination of
unknown protein samples.
• Comprehensive—13 purified isoforms from pI 3.5–10.7;
no additional high range or low range markers are required • Storage buffer: 10% glycerol containing bromophenol
blue (0.01%) and methyl red (0.01%)
• Versatile—can be used for both native and
denaturing conditions • Storage conditions: upon receipt store at –20°C
Learn more at
thermofisher.com/iefstandards Recommended products
The IEF Marker 3-10 is applicable to all IEF gels (vertical or horizontal).

Electrophoresis chambers
and power supplies
Electrophoresis run considerations:
In electrical terms, the process of

electrophoresis is closely
associated with the following
equations derived from Ohm’s Law:
Voltage = Current × Resistance (V = IR)
Wattage = Current × Voltage (W = IV)
Resistance Current
The electrical resistance of the assembled electrophoresis cell For a given gel/buffer system, at a given temperature, current
is dependent on buffer conductivity, gel thickness, temperature, varies in proportion to the field strength (voltage) and cross-
and the number of gels being run. Although the resistance is sectional area (thickness and number of gels). When using a
determined by the gel system, the resistance varies over the constant current setting, migration starts slow, and accelerates
course of the run. over time, thus favoring stacking in discontinuous gels.
• In discontinuous buffer systems (and to a lesser extent in When running under constant current, set a voltage limit on the
continuous buffer systems) resistance increases over the course power supply at, or slightly above the maximum expected voltage
of electrophoresis. This occurs in the Tris-glycine buffer system to avoid unsafe conditions. At constant current voltage increases
as highly conductive chloride ions in the gel are replaced by less as resistance increases. If a local fault condition occurs (e.g., a
conductive glycine ions from the running buffer. bad connection), high local resistance may cause the voltage to
reach the maximum for the power supply, leading to overheating
• Resistance decreases as the temperature increases.
and damage of the electrophoresis cell.
Voltage Power
The velocity of an ion in an electric field varies in proportion to the
Wattage measures the rate of energy conversion, which is
field strength (volts per unit distance). The higher the voltage, the
manifested as heat generated by the system. Using constant
faster an ion moves. For most applications, we recommend a
power ensures that the total amount of heat generated by the
constant voltage setting.
system remains constant throughout the run, but results in variable
• A constant voltage setting allows the current and power to mobility since voltage increases and current decreases over the
decrease over the course of electrophoresis, providing a safety course of the run. Constant power is typically used when using
margin in case of a break in the system. IEF strips. When using constant power, set the voltage limit slightly
above the maximum expected for the run. High local resistance
• The constant voltage setting does not need adjustment to can cause a large amount of heat to be generated over a small
account for differences in number or thickness of gels being distance, damaging the electrophoresis cell and gels.
electrophoresed.

Which electrophoresis chamber system is right for you?

Mini Gel Tank XCell SureLock Mini-Cell XCell4 SureLock Midi-Cell
Gel capacity Up to 2 minigels Up to 2 minigels (8 x 8 cm) Up to 4 midigels (8 x 13 cm)
Cell dimensions 32 x 11.5 x 16 cm 14 x 13 x 16 cm 21 x 19 x 16 cm

(L x W x H) (height with lid on) (height with lid on) (height with lid on)
Advantages • The Mini Gel Tank is versatile and • XCell II Blot Module is available for • Advanced apparatus for easier,
compatible with NuPAGE, Bolt, or semi-wet protein transfers more reliable electrophoresis with
Novex minigels. The unique tank • Instrument incorporates a gel midigels
design enables convenient side- tension wedge in place of the rear
by-side gel loading and enhanced wedge used on earlier models
viewing during use.
• Mini Blot Module is available for wet
protein transfers.
Learn more at
thermofisher.com/electrophoresischambers

Mini Gel Tank

One tank, 181 gels
The Mini Gel Tank is designed for more intuitive use
and greater convenience compared to traditional
electrophoresis tanks (Figure 24). The unique,
side-by-side tank design allows you to perform
electrophoresis of 1 or 2 minigels.
The Mini Gel Tank offers:

• Versatility—compatible with all of our minigels, including
NuPAGE, Novex, Bolt, and specialty gels
• Easy sample loading—forward-facing well configuration
• Simultaneous visualization of both gels—streamlined, Specifications

S
side-by-side tank configuration • Gel capacity: up to 2 minigels
• Simple monitoring of gels—white tank stand provides • Cell size (L x W x H): 32 x 11.5 x 16 cm (height with lid on)
easy visualization of prestained markers
• Buffer requirement: 400 mL for each minigel chamber
• Less running buffer required—gel chambers are
• Material: polycarbonate
separated, so you only need to load sufficient buffer for
each gel to the specified fill line • Chemical resistance: not compatible with acetone, chlorinated
hydrocarbons, or aromatic hydrocarbons
Watch our Mini Gel Tank video. thermofisher.com/minigeltank
Learn more at thermofisher.com/minigeltank

1. Snap the electrophoresis tank into the base, and 2. Remove the comb, and peel away the tape at the 3. Place the cassette in the chamber with the wells
place the cassette clamp(s) into the chamber(s) bottom of the gel cassette. facing towards you.
with the anode connector(s) (+) aligned to the center. Rinse the wells 3 times with 1X buffer. Hold the cassette in a raised position and close
Fill the chamber(s) with 1X buffer to the level of the clamp by moving the cam handle forward.
the cathode.
4. Make sure the wells are completely filled with 5. Hold the cassette and release the cassette clamp. 6. Make sure the power supply is off.
1X buffer.
Gently lower the casette so that it rests on the If only running one gel, remove the cassette
Load your samples and markers. bottom of the chamber, and close the cassette clamp. clamp from unused chamber.
Add 1X buffer to the level of the fill line. Place the lid on the tank and plug the
electrode cords into the power supply.
Turn the power supply on to begin electrophoresis.
Figure 24. How to use the Mini Gel Tank.
Figure 25. Electrophoresis of Bolt gel using the Mini Gel Tank. Protein
standards and samples were loaded at 10 μL sample volumes in an
Invitrogen™ Bolt™ 4–12% Bis-Tris Plus Gel. Electrophoresis was performed
using the Mini Gel Tank at 200 V (constant). Sharp, straight bands with
consistent migration patterns were observed after staining with SimplyBlue
SafeStain. Images were acquired using a flatbed scanner. Lane 1: SeeBlue
Plus2 Prestained Standard; Lane 2: 10 μg E. coli lysate; Lane 3: Mark12
Unstained Standard (blend of 12 purified proteins); Lane 4: 40 μg HeLa cell
lysate; Lane 5: 20 μg HeLa cell lysate; Lane 6: 5 μg BSA; Lane 7: 40 μg
Jurkat cell lysate; Lane 8: 5 μg GST fusion protein; Lane 9: Novex Sharp
Unstained Protein Standard; Lane 10: 5 μg β-galactosidase.
The Mini Blot Module is a wet transfer device that conveniently fits into
the chambers of the Mini Gel Tank to easily transfer proteins from minigels
to nitrocellulose or PVDF membranes.

XCell SureLock Mini-Cell

Simultaneous electrophoresis of up
to 2 minigels
The unique design of the Invitrogen™ XCell™

SureLock Mini-Cell allows you to run minigels
quickly and easily without any clamps or grease
(Figure 26). The tight seal provided by the gel
tension wedge results in leak-free, consistent
performance. The XCell SureLock Mini-Cell is
compatible with NuPAGE, Novex, and specialty
gels (Figure 27).
Key features of the XCell SureLock Mini-Cell: Specifications

• User-friendly design—uses single gel tension wedge with • Gel capacity: up to 2 minigels
no clamps or grease
• Cell size (L x W x H): 14 x 13 x 16 cm (height with lid on)
• Flexibility—perform electrophoresis of 2 minigels
• Buffer chamber requirement (Novex minigels):
simultaneously
– Upper buffer chamber: 200 mL
• Unique, heat dissipating design—no need for a
– Lower buffer chamber: 600 mL
cooling device
• Chemical resistance: The XCell SureLock Mini-Cell is
• Built-in safety features—retractable plugs, recessed jacks,
impervious to most alcohols but not compatible with acetone,
and a specially designed lid enhances user safety
chlorinated hydrocarbons (e.g., chloroform), or aromatic
hydrocarbons (e.g., toluene, benzene)
Learn more at thermofisher.com/surelockmini

Figure 26. How to use the

XCell SureLock Mini-Cell.
1. Drop buffer core into the lower 2. Lock the gel tension wedge in 3. Place the cell lid on the unit
buffer chamber of the XCell place, load samples, and fill the and you’re ready to run.
SureLock Mini-Cell. Insert one buffer chambers with the
minigel in front of the buffer core and appropriate running buffers.
a second minigel or the buffer dam
behind the buffer core.
Figure 27. Electrophoresis of NuPAGE Bis-Tris gels with

the XCell SureLock Mini-Cell. Lane 1: SeeBlue Plus2
Prestained Standard; Lane 2: 10 μg E. coli lysate; Lane 3:
Mark12 Unstained Standard (blend of 12 purified proteins);
Lane 4: 40 μg HeLa cell lysate; Lane 5: 20 μg HeLa cell lysate;
Lane 6: not used; Lane 7: 40 μg Jurkat cell lysate; Lane 8:
5 μg of a GST fusion protein; Lane 9: Invitrogen™ Novex™ Sharp
Protein Standard; and Lane 10: 5 μg β-galactosidase. Gel
electrophoresis was performed at 200 V (constant) and gels
were stained using SimplyBlue SafeStain. Images were acquired
using a flatbed scanner.
NuPAGE Bis-Tris gel in

XCell SureLock Mini-Cell
The XCell SureLock Mini-Cell can be easily adapted for transfer of proteins
from minigels to membranes by simply inserting the XCell II Blot Module
in the lower buffer chamber.

XCell4 SureLock Midi-Cell

Simultaneous electrophoresis of up to
4 midigels
The Invitrogen™ XCell4 SureLock™ Midi-Cell

allows simultaneous vertical electrophoresis of
1–4 midigels without leaking, enabling consistent
performance. The system is designed to dissipate
heat effectively and evenly, and enable high-
resolution results when using Novex midigels
(Figure 29).
Specifications
Key features of the XCell4 SureLock Midi-Cell: • Gel capacity: up to 4 midigels (8 x 13 cm)
• User-friendly design—leak-free electrophoresis without • Cell size (L x W x H): 21 x 19 x 16 cm (height with lid on)
clamps or grease
• Buffer chamber requirement:
• Flexibility—perform electrophoresis of 1–4 midigels – Upper buffer chamber: 175 mL x 4
• Unique, heat dissipating design—no need for a – Lower buffer chamber: 540–700 mL
cooling device • Chemical resistance: not compatible with acetone, chlorinated
hydrocarbons, or aromatic hydrocarbons
• Built-in safety features—specially designed lid enhances
user safety
Learn more at thermofisher.com/surelockmidi

A. 1. Insert the XCell4 SureLock Assembly in its unlocked 4. The upper buffer chamber (cathode) is the void
position into the center of the Midi-Cell base. The formed between a gel and the buffer core at the
XCell4 SureLock Assembly slides down over the center of each core.
protrusion in the Midi-Cell base.
2. Place one cassette on each side of the buffer core 5. Lock the XCell4 SureLock Assembly by moving the
for each of the two cores. For each cassette, the tension lever to the locked position (indicated on the
shorter “well” side of the cassette must face out XCell4 SureLock Assembly). This will squeeze the
towards the lower buffer chamber. gels and buffer cores together, creating leak-free
seals.
B. 3. While holding the assembly together with your 6. Proceed to loading samples and buffers.
hands (A), insert the buffer cores with the gel
cassettes into the lower buffer chamber such that
the negative electrode fits into the opening in the
gold plate on the lower buffer chamber (B). Always
hold the cassette assembly by its edges as shown
in the figure.
Note: If you are having difficulty inserting the
assembly into the lower buffer chamber, make sure
the cathode (black polarity indicator) of the buffer
core is aligned with the cathode (black polarity
indicator) of the lower buffer chamber.
Figure 28. How to use the XCell4 SureLock Midi-Cell with 4 gels.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 29. Quality of a precast NuPAGE Novex 4–12% Bis-Tris Midi
Gel with a variety of protein standards, lysates and purified proteins.
Electrophoresis was performed using MES running buffer and an XCell4
SureLock Midi Cell at 200 V (constant). Following electrophoresis, the
gel was stained using SimplyBlue SafeStain, destained using water, and
imaged using a flatbed scanner. Sharp, straight bands were observed.
Lanes 1, 10, 11, and 20 were each loaded with 5 μL of Mark12 Unstained
Standard (blend of 12 purified proteins). Lanes 2, 9, 12, and 19 were each
loaded with 10 μg of E. coli lysate. Lanes 3 and 18 were each loaded
with 6 μg of human IgG. Lanes 4 and 17 were each loaded with 6 μg of
human IgM. Lanes 5 and 16 were each loaded with 5 μL of SeeBlue Plus2
Prestained Protein Standard. Lanes 6 and 15 were each loaded with 5 μL
of BenchMark Protein Ladder. Lanes 7 and 14 were each loaded with
15 μL of MagicMark XP Western Protein Standard. Lanes 8 and 13 were
each loaded with 5 μL of HiMark Unstained Protein Standard.

PowerEase 90W
Power Supply
Simple, affordable power supply
specifically for minigel electrophoresis
The Invitrogen™ PowerEase™ 90W Power Supply

is designed specifically for minigel electrophoresis.
The straightforward, intuitive interface makes the
powering of gel runs a simple and easy process.
In addition, the PowerEase 90W Power Supply
features:
• Constant voltage or current settings
• Built-in timer for walk-away gel electrophoresis
• Output jacks that are compatible with most

electrophoresis devices
PowerEase 300W
Power Supply Learn more at thermofisher.com/powerease
Programmable power supply designed

for high-throughput gel electrophoresis
The Invitrogen™ PowerEase™ 300W Power Supply

is a fully programmable power supply designed
for high-throughput gel electrophoresis. The
straightforward, intuitive interface makes the
powering of gel runs a simple and easy process.
In addition, the PowerEase 300W Power Supply
features:
• Constant voltage, current, or power settings
• Built-in timer for walk-away gel electrophoresis
• Up to 10 custom programs with 10 steps each
• Four sets of output jacks that are compatible

with most electrophoresis devices

Run the gel

Table 4. Gel running conditions in electrophoresis chamber systems.
Running conditions in XCell Surelock Mini-Cell Running conditions in Mini Gel Tank
Starting End Approximate Starting Approximate
current current run time current End current run time
Voltage (V) (mA)* (mA)* (minutes) Voltage (V) (mA)* (mA)* (minutes)
Bolt 4–12% (MES) NA NA NA NA 200 160 70 20
Bolt 4–12% (MOPS) NA NA NA NA 200 160 50 35
NuPAGE 4–12% Bis-Tris 200 100 to 125 60 to 80 35 200 160 90 30
(MES)
NuPAGE 4–12% Bis-Tris 200 100 to 125 60 to 80 50 200 140 50 42
(MOPS)
Novex 4–20% Tris-Glycine 125 30 to 40 8 to 12 90 125 40 10 100
(denatured)
Novex 4–20% Tris-Glycine 125 6 to 12 3 to 6 1 to 12 125 30 10 90
(native) hours
NuPAGE 3–8% Tris-Acetate 150 40 to 55 25 to 40 60 150 60 20 50
(denatured)
NuPAGE 3–8% Tris-Acetate 150 18 7 2 to 3 hours 150 40 10 100
(native)
Novex 10–20% Tricine 125 80 40 90 125 110 40 65
NativePAGE 3–12% 150 12 to 16 2 to 4 90 to 115 150 10 <10 80
pH 3-10 IEF 100 7 NA 60 100 8 NA 60
200 NA NA 60 200 NA NA 60
500 NA 5 30 500 NA 5 30
10% Zymogram (Gelatin) 125 30 to 40 8 to 12 90 125 40 10 90
* Per gel.
Note: Run times may vary depending on the power supply and gel percentage.

Troubleshooting tips
XCell SureLock Mini-Cell troubleshooting
Observation Cause Solution
Run taking longer Buffers are too dilute Check if buffer was diluted properly. Check buffer recipe;
than usual dilute from concentrate or remake if necessary.
Upper buffer chamber is leaking Make sure the buffer core is firmly seated, the gaskets are in
place and the gel tension lever is locked.
Voltage is set too low Set correct voltage.
Current reading on power Tape left on the bottom of the cassette Remove tape from bottom of cassette.
supply is zero or very low
Connection to power supply Check all connections with a voltmeter for conductance.
not complete
Insufficient buffer level Make sure the upper buffer (cathode) is covering the wells of
the gel. Be sure there is sufficient buffer in the Lower Buffer
Chamber to cover the slot at the bottom of the gel.
Run is faster than normal with Buffers are too concentrated Check buffer recipe; dilute or re-make if necessary.
poor resolution or incorrect
Voltage, current, or wattage is set at a Decrease power conditions to recommended running

higher limit conditions (see page 59).
Cannot see the sample wells There is little contrast between the Mark cassette at the bottom of the wells with a marker pen
to load sample sample well and the rest of the gel prior to assembling the Upper Buffer Chamber. Illuminate
the bench area with a light source placed directly behind the
XCell SureLock unit.
Mini Gel Tank troubleshooting

Observation Cause Solution
Run taking longer than usual Buffers are too dilute Check buffer recipe; dilute from concentrate or remake
if necessary.
Buffer chamber is leaking Make sure the cassette clamp is firmly seated, the gaskets
are in place and the cassette clamp is locked.
Current is set too low Set correct current.
Current reading on power Tape left on the bottom of the cassette Remove tape from bottom of cassette.
supply is zero or very low
Connection to power supply Check all connections with a voltmeter for conductance.
not complete
Insufficient buffer level Make sure there is sufficient buffer in the electrophoresis tank
to cover the wells of the gel.
Run is faster than normal with Buffers are too concentrated Check buffer recipe; dilute or re-make if necessary.
poor resolution or incorrect
Current is set at a higher limit Decrease current to recommended running conditions (see
page 59).
Cannot see the sample wells There is little contrast between the Mark cassette at the bottom of the wells with a marker pen
to load sample sample well and the rest of the gel prior to placing the cassette in the electrophoresis tank.
Electrophoresis troubleshooting
Problem Possible cause Suggested solution
Run taking longer time with Running buffer too dilute Make fresh running buffer and use a 1X dilution.
recommended voltage
Current too high and Running buffer too concentrated Make fresh running buffer and use a 1X dilution.
excessive heat generated
with recommended voltage
Current too low or no current Incomplete circuit Remove the tape from the bottom of the gel cassette
with recommended voltage prior to electrophoresis. Make sure the buffer covers
sample wells; check the wire connections on the
buffer core.
Streaking of proteins Sample overload Load less protein.
High salt concentration in sample Decrease the sample salt concentration by dialysis or
gel filtration.
Sample precipitates Increase the concentration of SDS in the sample.
Contaminants such as lipids or DNA com- Centrifuge or clarify the sample to remove particulate
plexes in sample contaminants. Treat sample with nuclease(s).
Poorly poured gel Make sure the gel is poured evenly and all at once.
Fuzzy bands Protein sample only partially denatured Fully denature the protein.
Protein sample only partially reduced Make sure a sufficient amount of DTT or
β-mercaptoethanol is added.
Gel runs for too long Watch the dye front as an indicator for proper
running time.
Dumbbell shaped bands or Loading a large volume of sample causes Load appropriate volume of sample.
“smiling” bands incomplete stacking If the sample is too dilute, concentrate it using
ultrafiltration.
Uneven electric field during run Try to make sure the loading is symmetrical if the protein
concentration is known.
Uneven surface of the resolving gel Try to make the resolving gel surface even while pouring
the gel.
Expired gels Use the gels before the specified expiration date; Note:
NuPAGE gels have an extended 12 month shelf life,
minimizing the risk of having expired gels.

Stain the gel

Protein stains Silver stains
• Thermo Scientific™ Pierce™ Silver Stain
• Invitrogen™ SilverXpress™ Silver Stain

Once protein bands have been
• Thermo Scientific™ Pierce™ Silver Stain for Mass Spectrometry
separated by electrophoresis,
Fluorescent/specialty stains
they can be directly visualized • Invitrogen™ SYPRO™ Orange, Red, or Ruby gel stain
using different methods of in-gel • Pierce Reversible stain for nitrocellulose or PVDF membranes
• Pro-Q™ Emerald Glycoprotein stain

detection. Over the past several
• Pro-Q™ Diamond Phosphoprotein stain
decades, demands for improved
To visualize the proteins, a protein-specific, dye-binding or
sensitivity for small sample sizes color-producing chemical reaction must be performed on the
proteins within the gel. Depending on the particular chemistry
and compatibility with downstream of the stain, various steps are necessary to hold the proteins
in the matrix and to facilitate the necessary chemical reaction.
applications and detection Most staining methods involve some version of the same general
instrumentation have driven the incubation steps:
development of several basic DI H2O S DI H2O S
staining methods. Each method

1. Water wash. 2. Fix. 3. Water wash. 4. Stain. 5. Destain.
has particular advantages and

• A water-wash to remove electrophoresis buffers from the
disadvantages, and a number gel matrix
of specific formulations of each • An acid or alcohol wash to condition or fix the gel to limit
diffusion of protein bands from the matrix
type of method provide optimal • Treatment with the stain reagent to allow the dye or chemical to
diffuse into the gel and bind (or react with) the proteins
performance for various situations. • Destaining to remove excess dye from the background
gel matrix
Typically these stains can be classified broadly based on the dye
or molecule that helps visualize the protein stains: Depending on the particular staining method, two or more of
these functions can be accomplished with one step. For example,
Coomassie stains a dye reagent that is formulated in an acidic buffer can effectively
™
• Thermo Scientific PageBlue stain ™ fix and stain in one step. Conversely, certain functions require
several steps. For example, silver staining requires both a
• SimplyBlue SafeStain
staining reagent step and a developer step to produce the colored
• Thermo Scientific™ Imperial™ Protein Stain reaction product.
Learn more at thermofisher.com/proteinstains

Coomassie dye
protein gel stains
Convenient, ready-to-use reagents with
no permanent chemical modification
The most common methods for in-gel protein

detection use stains with Coomassie dye. These
stains use either the G-250 (colloidal) or R-250
form of the dye (Table 6). Colloidal Coomassie stain
can be formulated to effectively stain proteins within Our Coomassie stains provide sensitive protein detection along
one hour and require only water (no methanol or with simplified protocols. Example data and staining protocols are
acetic acid) for destaining. shown for SimplyBlue SafeStain (Figure 30, 33 and 34), PageBlue
Protein Staining Solution (Figure 32), and Imperial Protein Stain
Key features: (Figure 31 and 35).
• Simple—Coomassie dye–based formulations are easy to

formulate and are widely used Learn more at
thermofisher.com/coomassiestains
• Easy to use—simply soak the gel in stain solution and
destain to observe protein bands
• Economical—Coomassie dye–based stain formulations are

cost effective
• Flexible—useful for qualitative visualization, quantitative

densitometry, and gel excision and analysis by
mass spectrometry
Table 6. Coomassie dye–based protein gel stains.
PageBlue Protein
SimplyBlue SafeStain Imperial Protein Stain
Staining Solution
Type G-250 R-250 G-250
Limit of detection >7 ng 3 ng 5 ng
Time to stain (min) 12 60 60
Compatible with:
PVDF membranes Yes Yes Yes
Nitrocellulose membranes No No No
Reusable No No Yes (up to 3x)
Mass spectrometry Yes Yes Yes

compatible
Color Purple Purple Blue-green
Feature Free of methanol and acetic acid Photographs better than Free of methanol
Coomassie G-250 dye and acetic acid
Advantages Rapid, sensitive completely non-hazardous Fast, ultrasensitive Cost-effective
(does not require methanol or acetic acid protein detection option for fast,
fixatives or destains) staining sensitive staining

Protocols
S
DI H2O DI H2O DI H2O
Figure 30. SimplyBlue SafeStain protocol.
1. Wash the gel three 2. Add SimplyBlue 3. Wash gel with 100 mL 4. Additional water wash
times (5 minutes) with SafeStain (1 hour). of DI water for 1 hour. with 100 mL of DI water
ultrapure water. (1 hour) for increased
sensitivity.
S
DI H2O DI H2O
Figure 31. Imperial Protein Stain protocol.
1. Wash the gel three 2. Add Imperial Protein Stain 3. Water destain
times with deionized (5 minutes−1 hour). (15 minutes−overnight).
water (15 minutes).
DI H2O S DI H2O DI H2O
1. Wash the gel three 2. Add PageBlue Protein 3. Rinse gel two times 4. Wash gel one time Figure 32. PageBlue Protein Staining
times with ultrapure Staining Solution (1 hour). with ultrapure water with ultrapure water Solution protocol.
water (30 minutes). (<1 minute) (5 minutes)

Example data
1 2 3 4 5 6 7 8 9 10
2
3
1
Figure 33. Sensitive staining results with SimplyBlue SafeStain. The Figure 34. Two-dimensional electrophoresis (2DE) analysis of spinach
following samples were separated on a NuPAGE Novex 4-12% Bis-Tris chloroplast extract; staining with SimplyBlue SafeStain. Spinach
gel and then stained with SimplyBlue SafeStain. Lane 1: 6 μg protein mix; chloroplast extract was prefractionated in the ZOOM™ IEF Fractionator and
Lane 2: 1 μg rabbit IgG; Lane 3: 1 μg reduced BSA; Lane 4: 5 μg the individual fractions were then separated by 2DE using narrow pH range
E. coli lysate; Lane 5: 20 ng reduced BSA; Lane 6: 10 ng reduced BSA; ZOOM™ Strips and NuPAGE™ Novex 4–12% Bis-Tris ZOOM™ Gels. Gels were
Lane 7: 7 ng reduced BSA; Lane 8: 3 ng reduced BSA; Lane 9: 10 μL Coomassie stained using SimplyBlue SafeStain.
Mark12 Unstained Standard (blend of 12 purified proteins); Lane 10: 5 μL
Mark12 Unstained Standard.
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9
Rabbit IgG
BSA
Protein A
Protein G
Lysozyme
5-minute stain; 1-hour stain; 1-hour stain;
15-minute water destain 2-hour water destain overnight water destain
Figure 35. Enhanced sensitivity and clear background using Imperial Protein Stain. For even
greater sensitivity and reduced background, gels can be stained with Imperial Protein Stain for 1 hour
and washed with water from 1 hour to overnight. Lane 1: BSA only (6 μg); Lane 2–9: loaded left to right
with 1,000, 200, 100, 50, 25, 12, 6, and 3 ng protein sample.
Did you know?

Staining with a Coomassie stain
prior to silver staining allows for
Recommended products more uniform staining of certain
proteins since silver ions can
The Thermo Scientific Pierce Power Stainer is designed for rapid
interact with certain functional
Coomassie dye staining of proteins in up to two minigels and subsequent
groups such as carboxylic acid
removal of unbound stain from the gel in a single step. Refer to page 72 of
o
this brochure for more information.
groups, imidazole, sulfhydryls,
and amines.

Silver stains
Ultra-sensitive stains with optimized
protocols, manufactured for
minimal variability
Silver staining is the most sensitive colorimetric

method for detecting total protein, and functions
by the deposition of metallic silver at the location of
protein bands. Silver ions (from silver nitrate in the
stain reagent) interact and bind with certain protein
functional groups. The strongest interactions
occur with carboxylic acid groups (Asp and Glu),
imidazole (His), sulfhydryl groups (Cys), and amines
(Lys). Various sensitizer and enhancer reagents are
essential for controlling the specificity and efficiency We offer highly sensitive silver stains with short protocol times
of silver ion binding to proteins and effective that are also compatible with mass spectrometry (Table 7). The
conversion (development) of the bound silver to SilverXpress™ Silver Staining Kit provides nanogram-level sensitivity
metallic silver. with minimal background (Figure 37), while the Pierce™ Silver Stain
Kit provides protocol flexibility (Figure 38 and 39).
Key features:
• Sensitive—silver stains are highly sensitive stains that allow
Learn more at thermofisher.com/silverstains
for visualization of proteins at sub-nanogram levels
• Easy to use and flexible—optimized for minimal steps and

flexibility to accommodate shorter or longer protocols
• Workflow compatible—our mild chemical formulations

help ensure compatibility with mass spectrometry
and sequencing
• Robust performance—detailed protocol enables

consistent results with clear background

Table 7. Silver stain kits.
Pierce Silver Stain for SilverXpress Silver

Pierce Silver Stain Kit
Mass Spectrometry Staining Kit
Components (steps) 6 (17) 4 (15) 5 (13)
Time required 1 hr 13 min 2 hr 25 min 2 hr
Limit of detection 0.25 ng 0.25 ng 0.86 ng
Mass spectrometry Yes Yes Yes

compatible
Storage Room temperature Room temperature 4°C
Stability 1 year 1 year 6 months
Advantages • Fast and sensitive staining and destaining of • Rapid, ultrasensitive and • Nanogram-level
protein gels versatile silver stain system sensitivity for silver
• Optimized for peptide recovery after in-gel • Flexible gel fixation (30 min to staining with minimal
typsin digestion for mass spectrometry overnight) and staining (5 min to background
• Flexible gel fixation (15–30 min to overnight) 20 hours)
and staining (1–30 min)
Protocols and example data

F SZ
H2O
1 2 3 4 5 6 7 8 9 10
1. Wash the gel 2. Fix the gel in Fixing Solution 3. Decant the Fixing Solution
with water. for 10 minutes. and incubate the gel in 2
changes of Sensitizing Solution.
S
H 2O
4. Decant the Sensitizing Solution 5. Incubate the gel in Staining Solution.

and rinse the gel two times with Figure 37. Crystal clear background with the SilverXpress Kit. Samples
ultrapure water. were separated on a NuPAGE Novex 4-12% Bis-Tris gel and stained with
the SilverXpress Kit. Lanes 1, 10: Mark12 Unstained Standard (blend of 12
H2O D purified proteins) diluted 1:4; Lane 2: Mark12 Unstained Standard diluted
1:8; Lane 3: Mark12 Unstained Standard diluted 1:16; Lane 4: Mark12
Unstained Standard diluted 1:32; Lane 5: Mark12 Unstained Standard
D
diluted 1:64; Lane 6: 1.6 ng BSA; Lane 7: 0.8 ng BSA; Lane 8: E. coli
6. Decant the Staining Solution 7. Incubate the gel in Developing Solution. lysate diluted 1:20; Lane 9: E. coli lysate diluted 1:80.
and rinse the gel two times with
ultrapure water.
SS
H 2O
8. Add the Stopping Solution directly 9. Decant the Stopping Solution and wash
to the gel when the desired the gel three times with ultrapure water.
staining intensity is reached.
Figure 36. SilverXpress Silver Staining Kit protocol.

Acetic Lysate staining MW marker staining

EtOH Acid
F
H2O
1. Wash 2 x 5 minutes 2. Fix 2 x 15 minutes in EtOH/

with ultrapure water. acetic acid.
SZ
10% EtOH
SZ
3. Incubate 2 x 5 minutes 4. Mix Sensitizer. Sensitize for 2 minutes, 30 seconds 2 minutes, 30 seconds
with 10% EtOH. Wash. 1 minute. Wash 2 x 1 minute. Development Time
Figure 39. Pierce Silver Stain Kit exhibits excellent senstivity. In

S standard minigels, proteins are detectable at greater than 0.25 ng per
band or spot.
5. Mix Silver Stain. Stain for 5 minutes.

Wash 2 x 20 seconds.
5% Acetic Acid
D
6. Mix Developer. Develop for 2-3 minutes. 7. Remove developer.

Stop with 5% Acetic
Acid for 10 minutes.
Figure 38. Pierce Silver Stain Kit protocol.

Fluorescent protein gel stains

Rapid, highly sensitive fluorescent Learn more at
thermofisher.com/fluorescentstains
stains for total protein detection
after electrophoresis Recommended products
For optimal sensitivity with Polaroid™ film, SYPRO™ Photographic Filter
Fluorescent gel stains are designed for use in 1D is recommended.
and 2D PAGE and offer sensitivities similar to that
obtained with silver staining techniques. Invitrogen™
SYPRO™ protein stains are easy-to-use fluorescent
stains for the detection of proteins separated by
PAGE (Table 8). Stained proteins can be viewed
with a standard UV or blue-light transilluminator or
with a laser scanner.
Features:
• Simple—no destaining or timed steps required; minimal
hands-on time
• Quantitative—linear quantitation range over two orders of

magnitude with low protein-to-protein variability
• Highly sensitive—typically more sensitive than Coomassie

dye–based stains and equivalent to silver stains
Table 8. SYPRO protein stains.
SYPRO Ruby stain SYPRO Orange stain SYPRO Red stain
Limit of detection 0.25 ng 4–8 ng 4–8 ng
Stain and destain time 90 min microwave; 18 hr standard ~1 hr ~1 hr
Ex/Em 280 nm, 450/610 nm 300 nm, 470/510 nm 300 nm, 550/630 nm
Ease of use Ready to use Supplied as stock solution Supplied as stock solution
Compatible applications Mass spectrometry, IEF, 2D gels, Mass spectrometry, IEF, 2D gels, Mass spectrometry, IEF, 2D gels,
on-membrane staining on-membrane staining on-membrane staining

Specialty protein stains

Our specialty protein stains include in-gel Learn more at thermofisher.com/specialtystains
phosphoprotein and glycoprotein detection and
on-membrane reversible protein staining kits (Table 9).
Table 9. Specialty protein stains.
Pro-Q Emerald 488 Glycoprotein Pro-Q Emerald 300 Glycoprotein Gel Pro-Q Diamond Phosphoprotein Gel
Gel and Blot Stain Kit and Blot Stain Kit Staining Kit
Detects Glycoproteins Glycoproteins Phosphoproteins
Sensitivity 4 ng glycoprotein per band 0.5 ng glycoprotein per band 1–16 ng phosphoprotein per band
Stain and ~6 hr ~5 hr 4–5 hr

destain time
Ex/Em 510/520 nm 280/530 nm 555/580 nm
Advantages Selective staining of glycoproteins Selective staining of glycoproteins Selective staining of phosphoproteins

Stain the gel

Electrophoretic staining technology—Pierce Power Stainer
The Thermo Scientific Pierce Good to know
Power Stainer consists of a

How does electrostaining work?
Thermo Scientific™ Pierce™ Power Cathode (–)
Station with activated Staining Coomassie staining pad
Pre-run and pre-washed SDS-PAGE gel

Software and a Thermo Scientific™ Destaining pad
Pierce™ Power Stain Cassette. It Anode (+)
is designed for rapid Coomassie

staining and destaining of proteins Pierce Power Stain Cassette
in polyacrylamide gels. Traditional Cathode (–)
Staining pad
Coomassie staining techniques Gel
Destaining pad
require one hour to overnight Anode (+)
staining and destaining to achieve The significant reduction in protein staining time is
accomplished by utilizing inonic Power Stain Solution and
desired results. When used in Destain Solution to electrophoretically drive the negatively
charged Coomassie R-250 dye out of the top gel pad, through
conjunction with Thermo Scientific™ the polyacrylamide gel matrix and the bottom gel pad, and
Pierce™ Midi and Mini Gel Power toward the positively charged anode.
Staining Kits, the Pierce Power

Stainer is designed to provide
staining efficiency in as few as
6 minutes that is equivalent to, or
better than, traditional Coomassie
staining techniques. Watch our Pierce Power Stainer video.
thermofisher.com/powerstainer

Pierce Power Stainer

Rapid Coomassie dye staining and
destaining in approximately 10 minutes
The Thermo Scientific™ Pierce™ Power Stainer

is designed for rapid Coomassie dye staining of
proteins in polyacrylamide gels and subsequent
removal of unbound stains to give sharply stained
protein bands with minimal or no background.
The Pierce Power Stainer offers: Specifications

• Speed—Coomassie dye staining and destaining of proteins • Mode of transfer: semi-dry blotting
in about 10 minutes
• Gel compatibility: SDS-PAGE gels
• Convenience—simultaneously stain and destain 1–2
• Running dimension: horizontal
minigels or 1 midigel
• Platform: Pierce™ Power System
• Reliable performance—enables staining results that are
equivalent to traditional staining techniques
• Easy touch programming—intuitive LCD touch-screen

Pierce Power Stainer Conventional Coomassie stain
interface includes preprogrammed protocols
Total time: 11 minutes Total time: 230 minutes

to overnight
1. Wash gel 1 × 5 minutes in 1. Wash gel 3 × 10 minutes in water
water 2. Incubate gel in Coomassie
2. Power Stain/Destain gel, 6 stain solution* for 60 minutes
minutes 3. Wash gel 2 × 10 minutes in water
4. Destain gel in destaining solution** for
3 × 20 minutes
5. Incubate gel in water for 60 minutes
to overnight
*Coomassie stain solution: 45% methanol, 10% acetic acid, 0.25% Coomassie R-250
**Destain solution: 30% ethanol, 5% acetic acid
Figure 40. Pierce Power Stainer saves time and maintains sensitivity.
Learn more at thermofisher.com/powerstainer

The Pierce Power System can be used both for fast Coomassie dye
staining of protein gels and for rapid semi-dry transfer of proteins from gel
to membrane. The Pierce Power Stainer can be upgraded by adding the
Pierce™ Power Blot Cassette to make a fully functional Pierce Power
System with blotting and staining capabilities.
Did you know?

Conventional Coomassie
dye–based staining techniques
require 1 hour to overnight
incubation.
Thermo Scientific Pierce Thermo Scientific Pierce

Power Stainer Power Blotter

Western blotting
Transfer and Detection
After electrophoresis, the Horseradish peroxidase (HRP) or alkaline phosphatase
(AP) are the most popular enzymes conjugated to
separated proteins are transferred antibodies used in the western blot workflow. After
incubating the membrane with the detection antibody
or blotted onto a second matrix, or antibodies, if an enzyme-conjugated antibody
was utilized, an appropriate substrate (chromogenic
generally a nitrocellulose or or chemiluminescent) is added and that results in a
detectable product. A popular substrate of choice is
polyvinylidene difluoride (PVDF) a chemiluminescent substrate that, when combined
with the enzyme, produces light as a byproduct. With
membrane. Next, the membrane the chemiluminescent substrate, the light output can
be captured on film or CCD camera. In recent years
is blocked to minimize potential fluorescent detection became a popular alternative
nonspecific binding of antibodies to the enzymatic detection since it allows for more
quantitative data analysis. Fluorescent detection
to the surface of the membrane. utilizes dye-labeled primary antibodies or dye-
labeled secondary antibodies and the signal output is
captured on an appropriate imaging system. Whatever
Detailed procedures vary widely for the detection substrate is used, the intensity of the signal should
steps of the western blot workflow. One common correlate with the abundance of the antigen on the
variation involves direct vs. indirect detection methods. blotting membrane.
In both the direct and indirect detection methods,
the blocked membrane is probed with an antibody We offer a wide range of reagents, kits, equipment,
(primary antibody) specific to the protein of interest and antibodies to facilitate every step of western
(antigen). In direct detection techniques, this antibody blot analysis.
is enzyme conjugated or labeled with a fluorophore.
However, in indirect detection techniques, the blocked
membrane is probed first with an antibody (primary
antibody) which is specific to the antigen followed
by another antibody (secondary antibody) raised
against the host species of the primary antibody. This
secondary antibody is often enzyme conjugated or
labeled with a fluorophore. The direct method is not
widely used as most researchers prefer the indirect
detection method for a variety of reasons.
75 75
Protein gel electrophoresis technical handbook
Key products for western blot transfer:
Wet Semi-dry Dry
Mini Blot Module Thermo Scientific Pierce iBlot™ 2 Dry Blotting System
Power Blotter
Key products for western blot detection include:
Automated detection Manual detection
Blocking buffers
Wash buffers
Detergents
Enhancers
Substrates
Stripping buffers
X-ray film
iBind™ Flex Western Device
Learn more at thermofisher.com/western

76 Appendix
Protocol quick reference

QUICK REFERENCE
Bolt™ Mini Gels Bolt™ Mini Gels

Instructions for performing electrophoresis using Bolt ™ Mini Gels are described below.
Prepare gel *\[VWLU[OLNLSJHZZL[[LWV\JOHUKYLTV]L[OLJHZZL[[L
and tank 9LTV]L[OLNLSJVTIHUKYPUZL^LSSZ[PTLZ^P[O?9\UUPUN)\MMLY
Prepare Reagent Reduced Sample Non-reduced Sample 9LTV]L[OL[HWLJV]LYPUN[OLZSV[H[[OLSV^LYWVY[PVUVM[OLJHZZL[[L
samples Sample x μL x μL
Load 7YLMPSS[OLJOHTILY^P[O?9\UUPUN)\MMLY[V[OLSL]LSVM[OLJH[OVKL
Bolt ™ LDS Sample Buffer (4X) 10 μL 10 μL
Bolt ™ Reducing Agent (10X) 4 μL — samples 7SHJL[OLJHZZL[[LPU[OLJOHTILY^P[O[OL^LSSZMHJPUN[V^HYKZ`V\
Deionized Water to 26 μL to 30 μL /VSK[OLJHZZL[[LPUHYHPZLKWVZP[PVUHUKJSVZL[OLJHZZL[[LJSHTW
Total Volume 40 μL* 40 μL* -PSSHSS^LSSZ^P[O?9\UUPUN)\MMLY
Heat samples at 70˚C for 10 minutes. 3VHK`V\YZHTWSLZHUKTHYRLYZ
* Scale samples up or down by adjusting all volumes proportionally. /VSK[OLJHZZL[[LHUKYLSLHZL[OLJHZZL[[LJSHTW
.LU[S`SV^LY[OLJHZZL[[L[V[OLIV[[VTVM[OLJOHTILYHUKJSVZL[OL

Prepare 1X Each chamber of the tank requires 400 mL of 1X SDS Running Buffer (mix JHZZL[[LJSHTW
Buffer 20 mL of 20X Bolt ™ MES or MOPS SDS Running Buffer with 380 mL of
(KK?I\MMLY[V[OLSL]LSVM[OLMPSSSPUL
deionized water). The same buffer type must be used for both chambers.
1–4 5–7
Run Run Bolt™ Mini Gels at constant voltage (1 or 2 mini gels). Fill Line
conditions
Running Buffer Standard Run Run Time*
MES 200 V 22 min
MOPS 200 V 32 min Cathode Cathode
* Run times may vary depending upon gel type and power supply.
For research use only. Not for use in diagnostic procedures.
QUICK REFERENCE
NuPAGE Bis-Tris Mini Gels NuPAGE Tris-Acetate Mini Gels

Instructions for electrophoresis using the XCell SureLock Mini-Cell are described below.
Prepare Reagent Denatur i ng Sampl e* Nati ve Sampl e
Samples Sam pl e x μL x μL
Prepare Reagent Reduced Sampl e Non- r educed Sampl e NuPAGE LDS Sample Buffer (4X) 2.5 μL --
Samples Sam pl e x μL x μL Tris-Glycine Native Sample Buffer (2X) -- 5 μL
NuPAGE LDS Sample Buffer (4X) 2.5 μL 2.5 μL D ei on i z ed Water to 7.5 μ L to 5 μL
NuPAGE Reducing Agent (10X) 1 μL -- Tot al Vol u m e 10 μ L 10 μ L
Deionized Water to 6.5 μL to 7.5 μL Samples Heat samples at 70˚C for 10 minutes Do not heat
Tot al Vol u m e 10 μ L 10 μ L *For reduced samples, add NuPAGE Reducing Agent (10X) to 1X.
Heat samples at 70˚C for 10 minutes.
Prepare 1X Denaturing Samples: Add 50 mL 20X NuPAGE Tris-Acetate SDS Running
Buffer Buffer to 950 mL deionized water. Native Samples: Add 100 mL 10X Tris-
Prepare 1X Add 50 mL 20X NuPAGE MES or MOPS SDS Running Buffer to 950 mL
Glycine Native Running Buffer to 900 mL deionized water.
Buffer deionized water to prepare 1X SDS Running Buffer.
Load Sample Load the appropriate concentration of your protein sample on the gel.
Load Buffer Fill the Upper (200 mL) and Lower (600 mL) Buffer Chambers with the
Load Buffer Fill the Upper (200 mL) and Lower (600 mL) Buffer Chambers with the ap- appropriate 1X Running Buffer. For reduced samples, use 200 mL 1X
propriate 1X Running Buffer. For reduced samples, use 200 mL 1X Running Running Buffer with 500 μL NuPAGE Antioxidant in the Upper Buffer Chamber.
Buffer with 500 μL NuPAGE Antioxidant in the Upper Buffer Chamber.
Run Voltage: 150 V constant
Run Voltage: 200 V constant Run Time: 1 hour (Denaturing gel), 2–3 hours (Native gel)
Conditions
Conditions Run Time: 35 minutes (MES Buffer), 50 minutes (MOPS Buffer) Expected 40–55 mA/gel (start); 25–40 mA/gel (end) for denaturing gel
Expected Current: 100–125 mA/gel (start); 60–80 mA/gel (end) Current: 18 mA/gel (start); 7 mA/gel (end) for native gel
Intended Use: For research use only. Not for human or animal therapeutic or diagnostic use.
QUICK REFERENCE
NuPAGE Bis-Tris Midi Gels NuPAGE Tris-Acetate Midi Gels

Instructions for electrophoresis of Bis-Tris Gels using the XCell4 SureLock Midi-Cell are
described below. Prepare Reagent Denaturing Native Sample
Samples Sample
Prepare Sample x μL x μL
Reagent Reduced sample Non-reduced
Samples sample NuPAGE LDS Sample Buffer (4X) 2.5 μL —
Sample x μL x μL Tris-Glycine Native Sample Buffer (2X) — 5 μL
NuPAGE LDS Sample Buffer (4X) 2.5 μL 2.5 μL NuPAGE Reducing Agent (10X)* 1 μL —
NuPAGE Reducing Agent (10X) 1 μL — Deionized Water to 10 μL final to 10 μL final
Deionized Water to 10 μL final to 10 μL final *For reduced samples only.
Heat samples at 70˚C for 10 minutes. Heat denaturing samples at 70˚C for 10 minutes. Do not heat native samples.
Prepare Add 50 mL 20X NuPAGE MES or MOPS SDS Running Buffer to 950 mL Prepare Denaturing samples: Add 50 mL 20X NuPAGE Tris-Acetate SDS Running Buffer
1X Buffer deionized water to prepare 1X SDS Running Buffer. 1X Buffer to 950 mL deionized water. Native samples: Add 100 mL 10X Tris-Glycine
Native Running Buffer to 900 mL deionized water.
Add Buffer Fill Upper Buffer Chamber with 175 mL 1X NuPAGE SDS Running Buffer.
For reduced samples, use 175 mL 1X NuPAGE SDS Running Buffer with Add Buffer Fill Upper Buffer Chamber with 175 mL of the appropriate 1X Running Buffer.
435 μL NuPAGE Antioxidant in the Upper Buffer Chamber. Add a sufficient For reduced samples, use 175 mL 1X Running Buffer with 435 μL NuPAGE
volume of 1X NuPAGE SDS Running Buffer to the Lower Buffer Chamber. Antioxidant in the Upper Buffer Chamber. Add a sufficient volume of Running
Buffer the Lower Buffer Chamber.
Conditions Run Time: 40 min (MES Buffer), 55 min (MOPS Buffer) Run Voltage: 150 V constant
Expected Current: 160–200 mA/gel (start); 120–170 mA/gel (end) Conditions Run Time: 70 min (denaturing gel), 2–3 hours (native gel)
Expected Current: 70–90 mA/gel (start); 50–60 mA/gel (end); (denaturing gel)
40–45 mA/gel (start); 15–20 mA/gel (end); (native gel)
For Research Use Only. Not for use in diagnostic procedures.
QUICK REFERENCE
Novex Tris-Glycine Mini Gels Novex Tris-Glycine Mini Gels

Instructions are provided below for electrophoresis of NovexTris-Glycine Gels using the Non-Denaturing (Native) Electrophoresis
XCellSureLock Mini-Cell.
Prepare Reagent Sampl e
Denaturing Electrophoresis
Samples Sample x μL
Tris-Glycine Native Sample Buffer (2X) 5 μL
Prepare Reagent Reduced Sample Non-reduced Sample
Deionized Water to 5 μL
Samples Sample x μL x μL Total Volume 10 μL
Tris-Glycine SDS Sample Buffer (2X) 5 μL 5 μL
Do not heat samples for native electrophoresis.
NuPAGE Reducing Agent (10X) 1 μL --
Deionized Water to 4 μL to 5 μL
Prepare 1X Add 100 mL 10X Tris-Glycine Native Running Buffer to 900 mL deionized
Total Volume 10 μL 10 μL water to prepare 1X Tris-Glycine Native Running Buffer.
Buffer
Prepare 1X Add 100 mL 10X Novex Tris-Glycine SDS Running Buffer to 900 mL de-
Buffer ionized water to prepare 1X Tris-Glycine SDS Running Buffer. Load Buffer Fill the Upper Buffer Chamber with 200 mL and the Lower Buffer Chamber
with 600 mL of 1X Tris-Glycine Native Running Buffer.
Load Buffer Run Time: 1–12 hours
Fill the Upper Buffer Chamber with 200 mL and the Lower Buffer Chamber Conditions
with 600 mL of 1X Tris-Glycine SDS Running Buffer. Expected Current: 6–12 mA/gel (start); 3–6 mA/gel (end)
Run Voltage: 125 V constant Blot Gel For blotting denaturing and native gels, use 1X Tris-Glycine Transfer Buffer
Run Time: 90 minutes (dependent on gel percentage) with 20% methanol. Perform blotting at 25 V constant for 1–2 hours using
Conditions the XCell II Blot Module. The expected start current is 100 mA.
Expected Current: 30–40 mA/gel (start); 8–12 mA/gel (end)
Intended Use: For research use only. Not for human or animal therapeutic
or diagnostic use.
Appendix
78 Appendix
Protocol quick reference

QUICK REFERENCE
Tris-Glycine Midi Gels Non-denaturing (Native) Electrophoresis

Instructions for electrophoresis using the XCell4 SureLock Midi-Cell are described below.
Prepare Reagent Native Sample
Prepare Samples Sample x μL
Reagent Reduced sample Non-reduced
Samples sample Tris-Glycine Native Sample Buffer (2X) 5 μL
Sample x μL x μL Deionized Water to 10 μL final
Tris-Glycine SDS Sample Buffer (2X) 5 μL 5 μL Do not heat native samples.
NuPAGE Reducing Agent (10X) 1 μL —
Prepare Add 100 mL 10X Tris-Glycine SDS Running Buffer to 900 mL deionized water to
Deionized Water to 10 μL final to 10 μL final
1X Buffer prepare 1X Tris-Glycine SDS Running Buffer.
Prepare Add 100 mL 10X Tris-Glycine SDS Running Buffer to 900 mL deionized water to
1X Buffer prepare 1X Tris-Glycine SDS Running Buffer. Add Buffer Fill each Upper Buffer Chamber with 175 mL of 1X Tris-Glycine Native Running
Buffer. Fill the Lower Buffer Chamber up to the fill line mark with 1X Tris-
Load Sample Load the appropriate concentration of your protein sample on the gel. Glycine Native Running Buffer.
Add Buffer Fill each Upper Buffer Chamber with 175 mL 1X Tris-Glycine SDS Running Buffer. Run Voltage: 125 V constant
Fill the Lower Buffer Chamber up to the fill line mark with 1X Tris-Glycine Conditions Run Time: 1–12 hours
SDS Running Buffer. Expected Current: 35–40 mA/gel (start); 15–20 mA/gel
Voltage: 125 V constant
Run Run Time: 105 min (dependent on gel percentage)
Conditions Expected Current: 40–50 mA/gel (start); 20–25 mA/gel (end)
QUICK REFERENCE
NativePAGE™ Bis-Tris Gels Staining Protocol

A quick staining protocol for NativePAGE Gels using the Coomassie G-250 from the sample
Instructions are provided below for electrophoresis of NativePAGE™ Bis-Tris Gels using the XCell additive is described below. The total staining time is ~2–3 hours. Sensitivity is ~60 ng BSA.
SureLock Mini-Cell. Step Action Time
Place the gel in 100 mL fixing solution (40% methanol, 10% acetic 45
Prepare Reagent Sample with Detergent-free 1
acid) and microwave on high (950–1100 watts). seconds
Samples detergent sample
Sample x μL x μL
Shake the gel on an orbital shaker. 15
2
minutes
NativePAGE™ Sample Buffer (4X) 2.5 μL 2.5 μL
NativePAGE™ 5% G-250 Additive 0.25–1 μL* — 3 Discard fixing solution. —
Deionized Water to 10 μL to 10 μL Place the gel in 100 mL destain solution (8% acetic acid) and micro- 45
4
Do not heat samples for native gel electrophoresis. wave on high (950–1100 watts). seconds
*Ensure the final G-250 concentration is ¼th the detergent concentration. Shake the gel on an orbital shaker until the desired background is
5 —
obtained.
™ ™
Prepare 1X 1X NativePAGE Anode Buffer: Add 50 mL 20X NativePAGE Running Buffer
Running to 950 mL deionized water.
Buffer 1X NativePAGE™ Cathode Buffer: Add 50 mL 20X NativePAGE™ Running Buf-
fer and 50 mL 20X NativePAGE™ Cathode Additive to 900 mL deionized water.
Load Sample Fill wells with 1X NativePAGE™ Cathode Buffer and load samples prior to filling
the cathode chamber. Load an appropriate amount of your sample on the gel.
Add Buffer Fill the Upper Buffer Chamber with ~200 mL 1X NativePAGE™ Cathode Buffer.
Fill the Lower Buffer Chamber with ~550 mL 1X NativePAGE™ Anode Buffer.

Conditions Run Time: 90–115 min (3–12% gel); 105–120 min (4–16% gel)
Expected Current: 12–16 mA/gel (start); 2–4 mA/gel (end)

QUICK REFERENCE
Tricine Gels Tricine Gels

Instructions are provided below for electrophoresis of Tricine Gels using the XCell SureLock Blotting For blotting Tricine gels, use 1X Tris-Glycine Transfer Buffer with
Mini-Cell. Conditions 20% methanol. Perform transfer with nitrocellulose or PVDF membranes at
25 V constant for 1–2 hours using the XCell II Blot Module. The expected
start current is 100 mA.
Prepare Reagent Reduced Sampl e Non- r educed Sampl e
Samples Alternate The Tris-Glycine Transfer Buffer interferes with protein sequencing. If you
Sam pl e x μL x μL Transfer are performing protein sequencing, use 1X NuPAGE Transfer Buffer or
Tricine SDS Sample Buffer (2X) 5 μL 5 μL Buffers 0.5X TBE Transfer Buffer for blotting.
NuPAGE Reducing Agent (10X) 1 μL -- The NuPAGE Transfer Buffer protects against modification of the amino
Deionized Water to 4 μL to 5 μL acid side chains and is compatible with N-terminal protein sequencing
Tot al Vol u m e 10 μ L 10 μ L using Edman degradation.
Prepare 1X Add 100 mL 10X Novex Tricine SDS Running Buffer to 900 mL deionized
Buffer water to prepare 1X Tricine SDS Running Buffer.
Load Buffer Fill the Upper Buffer Chamber with 200 mL and the Lower Buffer Chamber
with 600 mL of 1X Tricine SDS Running Buffer.

Conditions Run Time: 90 minutes (dependent on gel percentage)
Expected Current: 80 mA/gel (start); 40 mA/gel (end)
For research use only. Not for human or animal therapeutic or diagnostic use.
QUICK REFERENCE
IEF Gels IEF Gels

Instructions are provided below for electrophoresis of IEF Gels using the XCell SureLock Prepare for 1. Stain and destain the IEF gel. Incubate the IEF gel in 100 mL 20% ethanol
Mini-Cell. 2D SDS/ for 10 minutes.
PAGE 2. Cut out the desired lane (strip) from the gel for transfer to a SDS gel.
Prepare Reagent Sample 3. Incubate the gel strip in 2 mL 2X SDS sample buffer and 0.5 mL ethanol for
Samples Sample x μL 3–5 minutes. Aspirate the sample buffer and rinse the gel strip with 1X SDS
IEF Sample Buffer pH 3–10 or pH 3–7 (2X) 5 μL Running Buffer.
Deionized Water to 10 μL final 4. Fill the SDS gel cassette with 1X SDS Running Buffer.
5. Trim the IEF gel strip to a length of 5.8–5.9 cm.
Prepare 1X IEF Anode Buffer: Add 20 mL 50X IEF Anode Buffer to 980 mL deion-
6a. Transfer the gel strip into a 1.0 mm SDS gel by sliding the strip into the
1X Buffer ized water. Chill to 4°C to 10°C.
2D-well using a gel loading tip. Avoid trapping air-bubbles between the strip
1X IEF Cathode Buffer: Add 20 mL IEF Cathode Buffer pH 3–10 (10X) or pH and the SDS gel. Wet a piece of thick filter paper (5.8 cm × 4 cm) in SDS
3–7 (10X) to 180 mL deionized water. Chill to 4°C to 10°C. Running Buffer and insert the long edge of the paper into the 2D-well to
push the gel strip into contact with the SDS gel.
Load Sample Load the appropriate concentration and volume of your protein on the gel.
6b. If transferring the gel strip into a 1.5 mm SDS gel, wet 2 pieces of thin filter
Fill the Upper Buffer Chamber with chilled 200 mL 1X IEF Cathode Buffer paper (5.8 cm × 4 cm) in 1X SDS Running Buffer. Sandwich the gel strip
Add Buffer
and the Lower Buffer Chamber with chilled 600 mL 1X IEF Anode Buffer. between the two filter papers along the long edge with the edge of the strip
protruding ~0.5 mm beyond the paper. Insert the sandwich into the 2D-well
Run Voltage: 100 V constant for 1 hour of the SDS gel without trapping air bubbles and push the strip down so it is
200 V constant for 1 hour in contact with the SDS gel.
Conditions
500 V constant for 30 minutes 7. Insert gel into the mini-cell, fill the buffer chambers with 1X SDS Running
Expected Current: 7 mA/gel (start); 5 mA/gel (end) Buffer, and perform SDS-PAGE.
8. After the dye front has moved into the stacking gel (~10 minutes),
Stain Gel Fix the IEF gel in 12% TCA or 12% TCA containing 3.5% sulfosalicylic acid disconnect power, remove the paper, and resume electrophoresis.
for 30 minutes. Stain the IEF gel with method of choice.
Appendix
80 Appendix
QUICK REFERENCE
Zymogram Gels Zymogram Gels
Instructions are provided below for electrophoresis of Zymogram Gels using the XCell Develop Gel 1. Dilute Zymogram Renaturing Buffer (10X) and Zymogram Developing Buffer
SureLock Mini-Cell. (10X) 1:9 with deionized water. Prepare 100 mL of each 1X buffer per 1–2
mini-gels.
Prepare 2. After electrophoresis, incubate the gel in 1X Zymogram Renaturing Buffer
Reagent Sample
Samples for 30 minutes at room temperature with gentle agitation.
Sample x μL
Tris-Glycine SDS Sample Buffer (2X) 5 μL 3. Decant Zymogram Renaturing Buffer and add 1X Zymogram Developing
Buffer to the gel.
Deionized Water to 10 μL final
4. Equilibrate the gel for 30 minutes at room temperature with gentle agitation.
Do not heat or reduce samples for Zymogram gels.
5. Decant buffer and add fresh 1X Zymogram Developing Buffer to the gel.
Prepare 1X Tris-Glycine SDS Running Buffer: Add 100 mL 10X Tris-Glycine SDS 6. Incubate the gel at 37°C for at least 4 hours or overnight for maximum
1X Buffer Running Buffer to 900 mL deionized water. sensitivity. Optimize results empirically by varying the sample load or
incubation time.
Load Sample Load the appropriate concentration and volume of your protein on the gel.
Stain Gel Zymogram (Blue Casein) 4–16% gels do not require staining. For non-pre-
Add Buffer Fill the Upper Buffer Chamber with 200 mL, and the Lower Buffer Chamber stained Zymogram gels, stain the gels with Colloidal Blue Staining Kit or
with 600 mL of 1X Tris-Glycine SDS Running Buffer. the SimplyBlue Safestain. Areas of protease activity appear as clear bands
against a dark background.
Run Time: 90 minutes (dependent on gel percentage) Sensitivity 10% Zymogram (Gelatin) Gel: 10-6 units of collagenase
Conditions
Expected Current: 30–40 mA/gel (start); 8–12 mA/gel (end) Level 12% Zymogram (Casein) Gel: 7 × 10-4 units of trypsin
4–16% Zymogram (Blue Casein) Gel: 1.5 × 10-3 units of trypsin
Ordering information
Product Quantity Cat. No. Product Quantity Cat. No.
Select precast gel Bolt Bis-Tris Plus Gels NuPAGE™ Novex™ 10% Bis-Tris Protein 1 box NP0307BOX
™
Bolt 8% Bis-Tris Plus Gels, 10-well 1 box NW00080BOX Gels, 1.0 mm, 9-well
Bolt™ 8% Bis-Tris Plus Gels, 12-well 1 box NW00082BOX NuPAGE™ Novex™ 10% Bis-Tris Protein 1 box NP0315BOX
Gels, 1.5 mm, 10-well
Bolt™ 8% Bis-Tris Plus Gels, 15-well 1 box NW00085BOX
NuPAGE™ Novex™ 10% Bis-Tris Protein 1 box NP0316BOX
Bolt™ 8% Bis-Tris Plus Gels, 17-well 1 box NW00087BOX Gels, 1.5 mm, 15-well
Bolt™ 10% Bis-Tris Plus Gels, 10-well 1 box NW00100BOX NuPAGE™ Novex™ 12% Bis-Tris Protein 1 box NP0344BOX
™
Bolt™ 10% Bis-Tris Plus Gels, 15-well 1 box NW00105BOX NuPAGE™ Novex™ 12% Bis-Tris Protein 10 gels NP0341BOX
™
Bolt 10% Bis-Tris Plus Gels, 17-well 1 box NW00107BOX
™
NuPAGE™ Novex™ 12% Bis-Tris Protein 2 gels NP0341PK2
Bolt 12% Bis-Tris Plus Gels, 10-well 1 box NW00120BOX
Bolt™ 12% Bis-Tris Plus Gels, 12-well 1 box NW00122BOX
NuPAGE™ Novex™ 12% Bis-Tris Protein 10 gels NP0342BOX
™
Bolt™ 12% Bis-Tris Plus Gels, 17-well 1 box NW00127BOX NuPAGE™ Novex™ 12% Bis-Tris Protein 2 gels NP0342PK2
™ Gels, 1.0 mm, 12-well
Bolt 4–12% Bis-Tris Plus Gels, 10-well 1 box NW04120BOX
™ NuPAGE™ Novex™ 12% Bis-Tris Protein 1 box NP0343BOX
Bolt 4–12% Bis-Tris Plus Gels, 12-well 1 box NW04122BOX
Bolt™ 4–12% Bis-Tris Plus Gels, 15-well 1 box NW04125BOX
NuPAGE™ Novex™ 12% Bis-Tris Protein 1 box NP0349BOX
™
Bolt 4–12% Bis-Tris Plus Gels, 17-well 1 box NW04127BOX Gels, 1.0 mm, 17-well
Bolt™ Empty Mini Gel Cassettes 20 cassettes NW2010 NuPAGE™ Novex™ 4–12% Bis-Tris 1 box NP0324BOX
™
Bolt Empty Mini Gel Cassette Combs, 20 combs NW3010 Protein Gels, 1.0 mm, 1-well
10-well NuPAGE™ Novex™ 4–12% Bis-Tris 10 gels NP0321BOX
Bolt™ Empty Mini Gel Cassette Combs, 20 combs NW3012 Protein Gels, 1.0 mm, 10-well
12-well NuPAGE™ Novex™ 4–12% Bis-Tris 2 gels NP0321PK2
™
Bolt Welcome Pack B, 4–12%, 15-well 1 kit** NW0412B Protein Gels, 1.0 mm, 10-well
Bolt™ Welcome Pack A, 4–12%, 10-well 1 kit** NW0412A NuPAGE™ Novex™ 4–12% Bis-Tris 10 gels NP0322BOX
Protein Gels, 1.0 mm, 12-well
One box contains 10 gels.
** Bolt Welcome Pack kit includes Mini Gel Tank, 2 boxes of Bolt 4–12% Gels NuPAGE™ Novex™ 4–12% Bis-Tris 2 gels NP0322PK2
(10-well/15-well), Bolt MES Running Buffer (20X), Bolt LDS Sample Buffer (4X), Bolt Protein Gels, 1.0 mm, 12-well
Sample Reducing Agent (10X) and SeeBlue™ Plus2 Prestained Standard
NuPAGE™ Novex™ 4–12% Bis-Tris 10 gels NP0323BOX
NuPAGE Bis-Tris Mini Gels (8 cm x 8 cm) Protein Gels, 1.0 mm, 15-well
NuPAGE™ Novex™ 10% Bis-Tris Protein 1 box NP0304BOX NuPAGE™ Novex™ 4–12% Bis-Tris 2 gels NP0323PK2
Gels, 1.0 mm, 1-well Protein Gels, 1.0 mm, 15-well
NuPAGE™ Novex™ 10% Bis-Tris Protein 10 gels NP0301BOX NuPAGE™ Novex™ 4–12% Bis-Tris 10 gels NP0329BOX
NuPAGE™ Novex™ 10% Bis-Tris Protein 2 gels NP0301PK2 NuPAGE™ Novex™ 4–12% Bis-Tris 2 gels NP0329PK2
NuPAGE™ Novex™ 10% Bis-Tris Protein 10 gels NP0302BOX NuPAGE™ Novex™ 4–12% Bis-Tris 1 box NP0327BOX
NuPAGE™ Novex™ 10% Bis-Tris Protein 2 gels NP0302PK2 NuPAGE™ Novex™ 4–12% Bis-Tris 10 gels NP0335BOX
NuPAGE™ Novex™ 10% Bis-Tris Protein 1 box NP0303BOX NuPAGE™ Novex™ 4–12% Bis-Tris 2 gels NP0335PK2
Appendix
82 Appendix
NuPAGE™ Novex™ 4–12% Bis-Tris 10 gels NP0336BOX NuPAGE™ Novex™ 3–8% Tris-Acetate 2 gels EA0375PK2
Protein Gels, 1.5 mm, 15-well Protein Gels, 1.0 mm, 10-well
NuPAGE™ Novex™ 4–12% Bis-Tris 2 gels NP0336PK2 NuPAGE™ Novex™ 3–8% Tris-Acetate 10 gels EA03752BOX
Protein Gels, 1.5 mm, 15-well Protein Gels, 1.0 mm, 12-well
NuPAGE Bis-Tris Midi Gels (8 cm x 13 cm) NuPAGE™ Novex™ 3–8% Tris-Acetate 2 gels EA03752PK2
™ ™
NuPAGE Novex 10% Bis-Tris Midi 1 box WG1201BOX Protein Gels, 1.0 mm, 12-well
Protein Gels, 12+2-well NuPAGE™ Novex™ 3–8% Tris-Acetate 1 box EA03755BOX
™ ™
NuPAGE Novex 10% Bis-Tris Midi 1 box WG1201A Protein Gels, 1.0 mm, 15-well
Protein Gels, 12+2-well, w/adapters NuPAGE™ Novex™ 3–8% Tris-Acetate 1 box EA0378BOX
NuPAGE™ Novex™ 10% Bis-Tris Midi 1 box WG1202BOX Protein Gels, 1.5 mm, 10-well
Protein Gels, 20-well NuPAGE™ Novex™ 3–8% Tris-Acetate 1 box EA03785BOX
™ ™
NuPAGE Novex 10% Bis-Tris Midi 1 box WG1202A Protein Gels, 1.5 mm, 15-well
Protein Gels, 20-well, w/adapters NuPAGE™ Novex™ 7% Tris-Acetate 1 box EA0355BOX
™ ™
NuPAGE Novex 10% Bis-Tris Midi 1 box WG1203BOX Protein Gels, 1.0 mm, 10-well
Protein Gels, 26-well NuPAGE™ Novex™ 7% Tris-Acetate 1 box EA03552BOX
NuPAGE™ Novex™ 10% Bis-Tris Midi 1 box WG1203A Protein Gels, 1.0 mm, 12-well
Protein Gels, 26-well, w/adapters NuPAGE™ Novex™ 7% Tris-Acetate 1 box EA03555BOX
™ ™
NuPAGE Novex 4–12% Bis-Tris Midi 1 box WG1401BOX Protein Gels, 1.0 mm, 15-well
Protein Gels, 12+2-well NuPAGE™ Novex™ 7% Tris-Acetate 1 box EA0358BOX
™ ™
NuPAGE Novex 4–12% Bis-Tris Midi 1 box WG1401A Protein Gels, 1.5 mm, 10-well
Protein Gels, 12+2-well, w/adapters NuPAGE™ Novex™ 7% Tris-Acetate 1 box EA03585BOX
™ ™
NuPAGE Novex 4–12% Bis-Tris Midi 1 box WG1402BOX Protein Gels, 1.5 mm, 15-well
Protein Gels, 20-well NuPAGE Tris-Acetate Midi Gels (8 cm x 8 cm)
NuPAGE™ Novex™ 4–12% Bis-Tris Midi 1 box WG1402A NuPAGE™ Novex™ 3–8% Tris-Acetate 1 box WG1601BOX
Protein Gels, 20-well, w/adapters Midi Protein Gels, 12+2W
NuPAGE™ Novex™ 4–12% Bis-Tris Midi 1 box WG1403BOX NuPAGE™ Novex™ 3–8% Tris-Acetate 1 box WG1601A
Protein Gels, 26-well Midi Protein Gels, 12+2W, w/adapters
NuPAGE™ Novex™ 4–12% Bis-Tris Midi 1 box WG1403A NuPAGE™ Novex™ 3–8% Tris-Acetate 1 box WG1602BOX
Protein Gels, 26-well, w/adapters Midi Protein Gels, 20W
NuPAGE™ Novex™ 8% Bis-Tris Midi 1 box WG1001BOX NuPAGE™ Novex™ 3–8% Tris-Acetate 1 box WG1602A
Protein Gels, 12+2-well Midi Protein Gels, 20W, w/adapters
NuPAGE™ Novex™ 8% Bis-Tris Midi 1 box WG1001A NuPAGE™ Novex™ 3–8% Tris-Acetate 1 box WG1603BOX
Protein Gels, 12+2-well, w/adapters Midi Protein Gels, 26W
NuPAGE™ Novex™ 8% Bis-Tris Midi 1 box WG1002BOX NuPAGE™ Novex™ 3–8% Tris-Acetate 1 box WG1603A
Protein Gels, 20-well Midi Protein Gels, 26W, w/adapters
NuPAGE™ Novex™ 8% Bis-Tris Midi 1 box WG1002A Novex Tris-Glycine Mini Gels (8 cm x 8 cm)
Protein Gels, 20-well, w/adapters Novex™ 10% Tris-Glycine Mini Protein 1 box EC6075BOX
™ ™
NuPAGE Novex 8% Bis-Tris Midi 1 box WG1003BOX Gels, 1.0 mm, 10-well
Protein Gels, 26-well Novex™ 10% Tris-Glycine Mini Protein 3 boxes EC6075BX30
™ ™
NuPAGE Novex 8% Bis-Tris Midi 1 box WG1003A Gels, 1.0 mm, 10-well - Value Pack
Protein Gels, 26-well, w/adapters Novex™ 10% Tris-Glycine Mini Protein 1 box EC60752BOX
NuPAGE Tris-Acetate Mini Gels (8 cm x 8 cm) Gels, 1.0 mm, 12-well
NuPAGE™ Novex™ 3–8% Tris-Acetate 10 gels EA0375BOX Novex™ 10% Tris-Glycine Mini Protein 1 box EC60755BOX
Protein Gels, 1.0 mm, 10-well Gels, 1.0 mm, 15-well
Novex™ 10% Tris-Glycine Mini Protein 1 box EC6078BOX Novex™ 18% Tris-Glycine Mini Protein 1 box EC6505BOX
Gels, 1.5 mm, 10-well Gels, 1.0 mm, 10-well
Novex™ 10–20% Tris-Glycine Mini 1 box EC6135BOX Novex™ 18% Tris-Glycine Mini Protein 1 box EC65055BOX
Novex™ 12% Tris-Glycine Mini Protein 1 box EC60055BOX Novex™ 4–12% Tris-Glycine Mini Protein 1 box EC6035BOX
Appendix
84 Appendix
Novex™ 6% Tris-Glycine Mini Protein 1 box EC60652BOX Novex™ 4–20% Tris-Glycine Midi 1 box WT4201A
Gels, 1.0 mm, 12-well Protein Gels, 12+2-well, w/adapters
Novex™ 6% Tris-Glycine Mini Protein 1 box EC60655BOX Novex™ 4–20% Tris-Glycine Midi 1 box WT4202BOX
Gels, 1.0 mm, 15-well Protein Gels, 20-well
Novex Tris-Glycine Midi Gels (8 cm x 13 cm) Novex™ 4–20% Tris-Glycine Midi 1 box WT4202A
™
Novex 10% Tris-Glycine Midi Protein 1 box WT0101BOX Protein Gels, 20-well, w/adapters
Gels, 12+2-well Novex™ 4–20% Tris-Glycine Midi 1 box WT4203BOX
™
Novex 10% Tris-Glycine Midi Protein 1 box WT0101A Protein Gels, 26-well
Gels, 12+2-well, w/adapters Novex™ 4–20% Tris-Glycine Midi 1 box WT4203A
Novex™ 10% Tris-Glycine Midi Protein 1 box WT0102BOX Protein Gels, 26-well, w/adapters
Gels, 20-well Novex™ 8% Tris-Glycine Midi Protein 1 box WT0081BOX
™
Novex 10% Tris-Glycine Midi Protein 1 box WT0102A Gels, 12+2-well
Gels, 20-well, w/adapters Novex™ 8% Tris-Glycine Midi Protein 1 box WT0081A
™
Novex 10% Tris-Glycine Midi Protein 1 box WT0103BOX Gels, 12+2-well, w/adapters
Gels, 26-well Novex™ 8% Tris-Glycine Midi Protein 1 box WT0082BOX
Novex™ 10% Tris-Glycine Midi Protein 1 box WT0103A Gels, 20-well
Gels, 26-well, w/adapters Novex™ 8% Tris-Glycine Midi Protein 1 box WT0082A
™
Novex 12% Tris-Glycine Midi Protein 1 box WT0121BOX Gels, 20-well, w/adapters
Gels, 12+2-well Novex™ 8% Tris-Glycine Midi Protein 1 box WT0083BOX
™
Novex 12% Tris-Glycine Midi Protein 1 box WT0121A Gels, 26-well
Gels, 12+2-well, w/adapters Novex™ 8% Tris-Glycine Midi Protein 1 box WT0083A
™
Novex 12% Tris-Glycine Midi Protein 1 box WT0122BOX Gels, 26-well, w/adapters
Gels, 20-well Novex™ 8–16% Tris-Glycine Midi Protein 1 box WT8161BOX
™
Novex 12% Tris-Glycine Midi Protein 1 box WT0122A Gels, 12+2-well
Gels, 20-well, w/adapters Novex™ 8–16% Tris-Glycine Midi Protein 1 box WT8161A
™
Novex 12% Tris-Glycine Midi Protein 1 box WT0123BOX Gels, 12+2-well, w/adapters
Gels, 26-well Novex™ 8–16% Tris-Glycine Midi Protein 1 box WT8162BOX
™
Novex 12% Tris-Glycine Midi Protein 1 box WT0123A Gels, 20-well
Gels, 26-well, w/adapters Novex™ 8–16% Tris-Glycine Midi Protein 1 box WT8162A
™
Novex 4–12% Tris-Glycine Midi Protein 1 box WT4121BOX Gels, 20-well, w/adapters
Gels, 12+2-well Novex™ 8–16% Tris-Glycine Midi Protein 1 box WT8163BOX
™
Novex 4–12% Tris-Glycine Midi Protein 1 box WT4121A Gels, 26-well
Gels, 12+2-well, w/adapters Novex™ 8–16% Tris-Glycine Midi Protein 1 box WT8163A
™
Novex 4–12% Tris-Glycine Midi Protein 1 box WT4122BOX Gels, 26-well, w/adapters
Gels, 20-well NativePAGE Gels
Novex™ 4–12% Tris-Glycine Midi Protein 1 box WT4122A NativePAGE™ Novex™ 3–12% Bis-Tris 1 box BN1001BOX
Gels, 20-well, w/adapters Protein Gels, 1.0 mm, 10-well
Novex™ 4–12% Tris-Glycine Midi Protein 1 box WT4123BOX NativePAGE™ Novex™ 3–12% Bis-Tris 1 box BN1003BOX
Gels, 26-well Protein Gels, 1.0 mm, 15-well
Novex™ 4–12% Tris-Glycine Midi Protein 1 box WT4123A NativePAGE™ Novex™ 4–16% Bis-Tris 1 box BN1002BOX
Gels, 26-well, w/adapters Protein Gels, 1.0 mm, 10-well
Novex™ 4–20% Tris-Glycine Midi 1 box WT4201BOX NativePAGE™ Novex™ 4–16% Bis-Tris 1 box BN1004BOX
Protein Gels, 12+2-well Protein Gels, 1.0 mm, 15-well
Novex Tricine Gels Prepare samples and select buffers: SDS-PAGE

™
Novex 10% Tricine Protein Gels, 1 box EC6675BOX Pierce™ SDS-PAGE Sample Prep Kit 50 89888
1.0 mm, 10-well reactions
Novex™ 10% Tricine Protein Gels, 1 box EC66752BOX Bolt Transfer Buffer (20X) 125 mL BT0006
1.0 mm, 12-well Bolt Transfer Buffer (20X) 1L BT00061
Novex™ 16% Tricine Protein Gels, 1 box EC6695BOX 4X Bolt LDS Sample Buffer 10 mL B0007
1.0 mm, 10-well
20X Bolt MES SDS Running Buffer 500 mL B0002
Novex™ 16% Tricine Protein Gels, 1 box EC66952BOX
1.0 mm, 12-well 20X Bolt MES SDS Running Buffer 5L B0002-02
Novex™ 16% Tricine Protein Gels, 1 box EC66955BOX 20X Bolt MOPS SDS Running Buffer 500 mL B0001
1.0 mm, 15-well 20X Bolt MOPS SDS Running Buffer 5L B0001-02
™
Novex 10–20% Tricine Protein Gels, 1 box EC6625BOX Bolt Antioxidant 15 mL BT0005
1.0 mm, 10-well
NuPAGE Tris-Acetate SDS Running 500 mL LA0041
Novex™ 10–20% Tricine Protein Gels, 1 box EC66252BOX Buffer (20X)
1.0 mm, 12-well
NuPAGE™ MOPS SDS Running Buffer 500 mL NP0001
Novex™ 10–20% Tricine Protein Gels, 1 box EC66255BOX (20X)
1.0 mm, 15-well
NuPAGE™ MOPS SDS Running Buffer 5L NP000102
Novex IEF Gels (20X)
Novex™ pH 3–7 IEF Protein Gels, 5 gels⁄box EC66452BOX NuPAGE™ MES SDS Running Buffer 5L NP000202
1.0 mm, 12-well (20X)
Novex™ pH 3–7 IEF Protein Gels, 5 gels⁄box EC6645BOX NuPAGE™ MES SDS Running Buffer 500 mL NP0002
1.0 mm, 10-well (20X)
Novex™ pH 3–10 IEF Protein Gels, 5 gels⁄box EC6655BOX Novex Tris-Glycine SDS Running Buffer 4x1L LC26754
1.0 mm, 10-well (10X)
Novex Zymogram Gels
Novex Tris-Glycine SDS Running Buffer 500 mL LC2675
Novex™ 12% Zymogram (Casein) 1 box EC64052BOX (10X)
Novex Tris-Glycine SDS Running Buffer 5L LC26755
Novex™ 4–16% Zymogram (Blue Casein) 1 box EC6415BOX (10X)
Novex™ Tricine SDS Running Buffer 500 mL LC1675
Novex™ 12% Zymogram (Casein) 1 box EC6405BOX (10X)
NuPAGE™ LDS Sample Buffer (4X) 10 mL NP0007
Novex™ 10% Zymogram (Gelatin) 1 box EC61755BOX ™
Novex Tricine SDS Sample Buffer (2X) 20 mL LC1676
Novex Tris-Glycine SDS Sample Buffer 20 mL LC2676
Novex™ 10% Zymogram (Gelatin) 1 box EC61752BOX
(2X)
Novex™ Tris-Glycine Transfer Buffer 500 mL LC3675
Novex™ 10% Zymogram (Gelatin) 1 box EC6175BOX
(25X)
E-PAGE™ High Throughput Gel System NuPAGE™ Transfer Buffer (20X) 125 mL NP0006
™
E-PAGE™ 8% Protein Gels, 48-well 8 gels EP04808 NuPAGE Transfer Buffer (20X) 1L NP00061
™
E-Holder™ Platform 2 units EH03 NuPAGE Antioxidant 15 mL NP0005
™
E-PAGE™ Loading Buffer 1 4.5 mL EPBUF01 Novex Tris-Glycine SDS Buffer Kit 1 kit LC2677
™
E-PAGE™ 6% Protein Gels, 96-well 8 gels EP09606 NuPAGE MOPS SDS Buffer Kit 1 kit NP0050
(for Bis-Tris Gels)
Daughter E-Base™ Device 1 unit EBD03
NuPAGE™ MES SDS Buffer Kit 1 kit NP0060
™
Mother E-Base Device 1 unit EBM03 (for Bis-Tris Gels)
Appendix
86 Appendix
NuPAGE™ Tris-Acetate SDS Buffer Kit 1 kit LA0050 Select Protein Standards: Unstained
(for Tris-Acetate gels), Contains 1 ea. PageRuler™ Unstained Low Range 2 x 250 μL 26632
LA0041, NP0004, NP0005, NP0007 Protein Ladder
Novex™ Tricine SDS Buffer Kit, 1 kit LC1677 PageRuler™ Unstained Protein Ladder 2 x 250 μL 26614
includes LC1676 & LC1675
NativeMark Unstained Protein Standard 5 x 50 μL LC0725
Pierce LDS Sample Buffer, Non- 5 mL 84788
Prestained
Reducing (4X)
PageRuler™ Prestained Protein Ladder 2 x 250 μL 26616
Pierce Lane Marker Non-Reducing 5 mL 39001
Sample Buffer PageRuler™ Prestained Protein Ladder 10 x 250 μL 26617
™
Pierce 10X Tris-Glycine SDS Buffer 1L 28362 PageRuler Plus Prestained 2 x 250 μL 26619
Protein Ladder
BupH™ Tris-Glycine-SDS Buffer Packs 40 packs 28378
PageRuler™ Plus Prestained 10 x 250 μL 26620
Native Electrophoresis
Protein Ladder
Novex Tris-Glycine Native Running 500 mL LC2672
Spectra™ Multicolor Broad Range 2 x 250 μL 26634
Buffer (10X)
Protein Ladder
Novex Tris-Glycine Native Sample 20 mL LC2673
Spectra™ Multicolor Broad Range 10 x 250 μL 26623
Buffer (2X)
Protein Ladder
NativePAGE™ Running Buffer (20X) 1L BN2001
HiMark™ Prestained Protein Standard 250 μL LC5699
NativePAGE™ Running Buffer Kit 1 kit BN2007 ™
Spectra Multicolor High Range 2 x 250 μL 26625
NativePAGE™ Cathode Buffer Additive 250 mL BN2002 Protein Ladder
(20X)
Western
NativePAGE™ Sample Buffer (4X) 10 mL BN2003
MagicMark™ XP Western 250 μL LC5602
™
NativePAGE 5% G-250 Sample 0.5 mL BN2004 Protein Standard
Additive
MagicMark™ XP Western 50 μL LC5603
™
NativePAGE Sample Prep Kit 1 kit BN2008 Protein Standard
DDM (n-dodecyl β-D-maltoside) (10%) 1 mL BN2005 Specialty
Digitonin (5%) 1 mL BN2006 PageRuler™ Prestained NIR 2 x 250 μL 26635

Protein Ladder
Zymography
BenchMark™ Fluorescent 125 μL LC5928
Novex™ Zymogram Developing Buffer 500 mL LC2671
Protein Standard
(10X)
BenchMark™ His-tagged 125 μL LC5606
Novex™ Zymogram Renaturing Buffer 500 mL LC2670
Protein Standard
(10X)
IEF Marker 3–10 500 μL 39212-01
IEF
Novex™ IEF Anode Buffer (50X) 100 mL LC5300
™
Novex IEF Cathode Buffer pH 3-10 (10X) 125 mL LC5310
™
Novex IEF Cathode Buffer pH 3-7 (10X) 125 mL LC5370
™
Novex pH 3-10 IEF Buffer Kit, 1 kit LC5317
Includes LC5300, LC5310, LC5311
Novex™ pH 3-7 IEF Buffer Kit, 1 kit LC5377
Includes LC5300, LC5370, LC5371
Novex™ IEF Sample Buffer pH 3-10 (2X) 25 mL LC5311
™
Novex IEF Sample Buffer pH 3-7 (2X) 25 mL LC5371
Electrophoresis chamber systems and power supplies Coomassie stains

Mini Gel Tank 1 unit A25977 PageBlue Protein Staining Solution 1L 24620
XCell SureLock Mini-Cell 1 unit EI0001 SimplyBlue SafeStain 1L LC6060
XCell4 SureLock™ Midi-Cell 1 each WR0100 SimplyBlue SafeStain 3.5 L LC6065

™
PowerEase™ 90W Power Supply 1 each PS0090 Imperial Protein Stain 1L 24615
(115 VAC) ™
Imperial Protein Stain 3x1L 24617
PowerEase™ 90W Power Supply 1 each PS0091 Silver stains
(230 VAC)
Pierce™ Silver Stain Kit 1L 24612
PowerEase™ 300W Power Supply 1 each PS0300
SilverXpress™ Silver Staining Kit 1 kit* LC6100
(115 VAC)
Pierce™ Silver Stain for 1L 24600
PowerEase™ 300W Power Supply 1 each PS0301
Mass Spectrometry
(230 VAC)
*1 kit contains sufficient reagents to stain 25 mini gels
Fluorescent and specialty stains

SYPRO™ Orange Protein Gel Stain 500 μL S-6650
SYPRO™ Orange Protein Gel Stain 10 x 50 μL S-6651
SYPRO™ Red Protein Gel Stain 500 μL S-6653
SYPRO™ Red Protein Gel Stain 10 x 50 μL S-6654
™
SYPRO Ruby Protein Gel Stain 1L S-12000
™
SYPRO Ruby Protein Gel Stain 200 mL S-12001
™
SYPRO Ruby Protein Gel Stain 5L S-21900
™
Pro-Q Emerald 488 Glycoprotein 1 kit P-21875
Gel Stain Kit
Pro-Q™ Diamond Phosophoprotein 1L P-33300
Gel Stain Kit
Pro-Q™ Diamond Phosophoprotein 200 mL P-33301
Gel Stain Kit
Pro-Q™ Diamond Phosophoprotein 5L P-33302
Gel Stain Kit
Pierce Power Stainer
Pierce Power Stainer 1 unit 22833
™
Pierce Power Stainer Welcome Pack 1 unit 22833SPCL*
*Welcome pack includes Pierce Power Station, Pierce Power Stain Cassette, Western Blot
Roller, Power Cord with C/13 Connector, Quick Start Guide, Pierce Mini Gel Power
Staining Kit
Appendix
Crossword puzzle challenge
To participate in the crossword puzzle challenge,
go to thermofisher.com/pagecrossword
1 2
6 7
10
11
12 13 14
15
C t d Th T h C tC dM k
A cross Down
5. Can you name one of the scientists who developed 1. Which tank is compatible with >180 mini gels? pg. 52 2
blue native polyacryr lamide gel electro
r phore
r sis? pg. 17 2. Which gel chemistryr minimizes pro r tein degra
r dation? pg. 9
6. Which tank is compatible with midigels? pg. 56 3. Who firs
r t published SDS-PAGE as a method for the
9. Which ladder can be used for accura r te molecular analysis of cleavage of structurar l pro
r teins in
weight estimation direr ctly on western blots? pg. 46 bacteriophage? pg. 7
10. Can you name one of the scientists who filed a 4. Can you name one of the scientists who firs r t
r l-pH Bis-Tris system in 1996? pg. 11
patent for the netura 1 described the theoryr of separa
r tion of amphoteric
12. Which gel has a unique wedge well design that pro r dient in the 1960s? pg. 21
r teins along a pH gra
allows you to load 2x the sample volume? pg. 10 7. What power supply is available for use with the Mini
13. Which pror tein ladder would you use for Gel Tank? pg. 58
r ximate determination of molecular weight? pg. 35
appro 8. What is a fast alternative to tra
r ditional Coomassie
15. Who won the Nobel Prize for analysis of serum staining? pg. 72
pro
r teins by electro
r phorer sis in 1948? pg. 5 11. Which ladder would you use for pre r cise
determination of molecular weight? pg. 35
14. Which type of gel has been re r fere
r nced in >20,000
publications? pg. 12
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are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Polaroid is a trademark of Polaroid
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Protein Gel Electrophoresis

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Western blotting

Protein gel electrophoresis

For a complete listing of all available products

Select precast gel

Prepare samples and select buffers

Select the standard

Choose the electrophoresis chamber system and power supply

Run the gel

Stain the gel

Sample preparation and Electrophoresis chamber Electrophoresis

on the following factors: • Electrophoresis conditions

The acrylamide matrix Electrophoresis conditions

Sample preparation and Electrophoresis chamber Electrophoresis

With the Tris-acetate system (Figure 3), three ions are

The combination of a lower-pH gel buffer (pH 6.4) and running

Select precast gel

Learn more at thermoﬁsher.com/proteingels

• NuPAGE Bis-Tris gels • Novex Tricine gels

Sample preparation and Electrophoresis chamber Electrophoresis

Low throughput Medium or high throughput

Coomassie dye High-sensitivity Downstream applications Large sample volume for

NuPAGE NuPAGE NuPAGE Bolt Bis-Tris

Bolt Bis-Tris Bolt Bis-Tris Bolt Bis-Tris

Molecular weight Isoelectric point Protease activity

1st Novex Tris-Glycine Novex Zymogram

NuPAGE Bis-Tris NuPAGE Bis-Tris

For ordering information refer to pages 81–87.

Choose the right gel chemistry for your research needs

The most widely used gel system for separating

• Cleavage of Asp-Pro bonds of proteins when heated at

Choosing the right gel percentage Choosing a well format

Sample preparation and Electrophoresis chamber Electrophoresis

Bolt Bis-Tris Plus mini gels

Invitrogen™ Bolt™ Bis-Tris Plus gels are precast

• Superior band quality and band volume—

 Learn more at thermoﬁsher.com/bolt

Bolt Bis-Tris Plus gels

“The new Bolt system is wonderful. I am

Did you know?

The Bolt Welcome Pack

Sample preparation and Electrophoresis chamber Electrophoresis

The Invitrogen™ NuPAGE™ SDS-PAGE gel system

• Average run time: ~35 minutes

• Superior protein band resolution and stability— • Gel dimensions:

• Fast sample run times—typically 35–50 minutes

• Long product shelf life—stable for 8–16 months ts acquired w

Figure 8. NuPAGE Bis-Tris and Tris-Acetate

A. NuPAGE Bis-Tris gels B. NuPAGE Tris-Acetate gels C. NuPAGE Tris-Acetate gels

Sample preparation and Electrophoresis chamber Electrophoresis

Novex Tris-Glycine gels

Invitrogen™ Novex™ Tris-Glycine gels are based on Speciﬁcations

Figure 10. Novex Tris-Glycine

 Learn more at thermoﬁsher.com/trisglycine

Novex Tris-Glycine gels

A. Denaturing separation B. Native separation††

Large proteins* Mid-size proteins† Small proteins† Wide range†

260 kDa 160 kDa

Sample preparation and Electrophoresis chamber Electrophoresis

The Invitrogen™ NativePAGE™ Bis-Tris gel system

For a complete listing of all available products

Learn more at thermoﬁsher.com/proteingels

Learn more at thermoﬁsher.com/bolt

Learn more at thermoﬁsher.com/trisglycine

Learn more at thermoﬁsher.com/nativepage

• Minimized protein modiﬁcation due to the lower pH of

Learn more at thermoﬁsher.com/tricine

Learn more at thermoﬁsher.com/ief

Learn more at thermoﬁsher.com/zymogram

Learn more at thermoﬁsher.com/epage

Learn more at 2 M Guanidinium•HCl 70%