Phos-tag™ SDS-PAGE GUIDEBOOK

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TM
 INDEX
  1．Principle and Application P3
  2．Phos-tag™ SDS-PAGE is… P4
  3．Protocol P6
     ［Ⅰ］Mn2+-Phos-tag™ SDS-PAGE P6
     ［Ⅱ］Zn2+-Phos-tag™ SDS-PAGE P11
  4．Trouble Shooting P14
  5．Optimization of Phos-tag™ SDS-PAGE Condition P17
  6．Application Data and References P18
  7．FAQ (Phos-tag™ Series) P22
  8．SuperSep Phos-tag™ P26
  9. Phos-tag™ Series P27
10. Related Products（Electrophoresis/Western Blotting) P29

Phos-tag™ Applications
Multiple possibilities of Phos-tag™
0U]P[YV 0U]P]V
Expressed Proteins Cells Animal tissues
Identification of
new phosphoproteins
Analysis of phosphorylation
status of endogenous proteins

Increase / Decrease
Kinase Assay Analysis of genetically
of phosphorylation
modified mice
by stimulation

Analysis of Kinase catalyzed No need for preparation of

signaling cascades. Anti phospho-Antibodies

No need for applying radioactivity

Ready for research of phosphorylation at various stages and for various purposes

−２−
 1．Principle and Application
1．Principle and Application

■ Phos-tag™ is …
Phos-tag™ is a functional molecule which captures phosphorylated Ser/Thr/Tyr and His/Asp/Lys. It is a synthesized chemical
compound designed from an alkaline phosphatase catalytic domain as a model. A series of reagents utilizing Phos-tag™ can
be used in the separation, detection, mass spectrometry analysis, and purification of phosphorylated proteins.

【Basic Structure of Phos-tag™】 Product Purpose of Use

Separation ： Separation is possible by SDS-PAGE
㻾 Phos-tag™ Acrylamide depending on the degree of phos-
㻻 㻻
phorylation.
㻼 㻻㻙
㻻
㻙 㻺 Separation ： Ready-to-use precast gel containing
㻺 SuperSep Phos-tag™
㻹 㻞㻗 50 μM Phos-tag™ Acrylamide
㻹㻞㻗 㻻㻙 㻺
㻺 㻺 㻺 Detection ： A substitute for the anti-phospho
Phos-tag™ Biotin
antibody used in western blot.

䡚䠍㻌㼚㼙 Phos-tag™ Agarose Purification ： Phosphorylated proteins are purified

by column chromatography.
M2+：Zinc ion or manganese ion
◇ Selectivity of binding of a phosphate ion Phos-tag™ Tip Purification ： Ready-to-use tip to purify phosphory-
(2-) is much higher than that of other lated peptides

anions. Analysis ： This is used in MALDI-TOF/MS analysis

Phos-tag™
◇ Stable complex is formed under physi- to improve the detection sensitivity of
Mass Analytical Kit
ological conditions (pH 5 to 8). phosphorylated molecules.
Phos-tag™ was developed by the Department of Functional Molecular Science at
Hiroshima University. http://www.phos-tag.com/
■ Principle
Non-phosphorylated protein
Two metallic ions cooperate Phosphorylated protein
to bind a phosphate group

TM
M

1. Divalent
D metal ions trap phosphorylated proteins during migration.
Phosphorylated forms can 2. Th
The higher the amount of phosphorylation, the slower the migration
be separated by the amount and velocity.
ve
site of phosphorylation. Se
3. Separation occurs based on phosphorylation levels.
(S
(Separation can occur if the phosphorylation sites are different,
ev
even with identical levels of phosphorylation)

■ Application ― Time-course of α-casein dephosphorylation ―

Phos-tag™ SDS-PAGE and conventional SDS-PAGE was used to separate α-casein samples, which have been
dephosphorylated over time (incubation duration: 0‒120 min) by alkaline phosphatase.
Phosphatase-
0 120 0 120
treated time

Phosphorylated α-casein

Phosphorylated α-casein
+
Non-phosphorylated Non-phosphorylated α-casein
α-casein
Conventional SDS-PAGE Phos-tag™ SDS-PAGE
10 % Acrylamide 10 % Acrylamide
100 μM Mn2+-Phos-tagTM Acrylamide

−３−
 2．Phos-tag™ SDS-PAGE is …
■ Phos-tag™ SDS-PAGE
Phos-tag™ SDS-PAGE is an electrophoresis technique capable of separating phosphorylated and non-phosphorylated
forms based on phosphorylation levels. Various stains, Western blotting (WB) and mass spectrometry (MS) can be
employed after electrophoresis. Phos-tag™ SDS-PAGE gels can be produced by adding a divalent metal (MnCl2 or
ZnCl2) and Phos-tag™ Acrylamide (Phos-tag™ molecule bound to acrylamide) to the SDS-PAGE resolving gel.

■ Features
◆ It can be used regardless of the type and position of amino acid residues.
  ・It can be used in phosphorylation analysis of unknown phosphoproteins.
  ・It can be used in the detection of new phosphorylation sites.

◆ Phosphorylated forms with different amounts and location of phosphorylation sites can
be separated.
  ・Able to determine the level of phosphorylation, as well as the amount of phosphorylated forms.

◆ Capable of simultaneously detecting phosphorylated and non-phosphorylated forms.

  ・Able to quantify various phosphorylated forms.
  ・Easily determine the presence of phosphorylation

◆ Radioisotopes or special equipment is not necessary (can be used immediately if there

are SDS-PAGE reagents and equipment)
  ・Experiments can be carried out easily and at low costs.

◆ After electrophoresis, WB- and MS-based analysis, as well as 2D gel electrophoresis

can be performed.
  ・WB： internal control protein analysis is possible. (See Application Data 3 and 4 on page #20.)
  ・MS：phosphorylation site combinations of various phosphorylated forms can be determined. (See Application
Data 1 and 2 on page #19.)
  ・2D gel electrophoresis：phosphorylated forms with identical isoelectric points or molecular weights can be
separated. (See Application Data 2 on page #19.)

■ Development from Phos-tag™ SDS-PAGE

By the combination of Phos-tag™ SDS-PAGE with various analysis methods, new information of phosphorylated
proteins can be obtained.

Western blotting MC* ‒ ‒ + + * MC：Microcystin

ATP ‒ + ‒ + （a phosphatase inhibitor）
Easy-recognizable phosphorylation Phosphorylated
〈Laemmli method〉
of your target proteins p35 SDS-PAGE
+
Simultaneous detection of Non-phosphorylated
phosphorylated/non-phosphorylated p35 〈Phos-tag™ SDS-PAGE〉
proteins with a general antibody
by their band shift differences.
No need to prepare an antiphos- Sample：Rat brain extract
pho antibody. Detection：Anti p35
Phosphorylated Lane 1：brain extract before incubation.
Applicable to analysis of p35 Lane 2-5：Incubate with (+) and
phosphorylation of endogenous without (-) MC or ATP
proteins.
Non-phosphorylated
p35
1 2 3 4 5

Mass Analysis 2D Electrophoresis

Phosphorylated forms with the same
By separating phosphorylated forms,
isoelectric point (same number of
each phosphorylation site combination
phosphorylation sites) can be separated.
can be detected.
Application Data → See the page #18

−４−
■ Application using Phos-tag™ SDS-PAGE

2．Phos-tag™ SDS-PAGE is …
See the page #29∼30 on the related products.

Objective Sample type Application Related Products Application Data

Lysate Ala substitute + WB ImmunoStar Series p.19 ④

To identify phosphorylation
Recombinant + MS
sites Silver Stain MS Kit,
Purified protein Immunoprecipitation p.18 ①, ②
nanoLC-MS/MS
+ MS

Lysate WB ImmunoStar Series p.19 ④、p.20 ⑤, ⑥

To simultaneously detect
and quantify various CBB staining, Silver Quick CBB Plus,
phosphorylated forms Purified protein p.18 ①, ②
staining, etc. Silver Stain MS Kit, etc.

To confirm presence of
Lysate WB ImmunoStar Series p.19 ③、p.20 ⑤, ⑥
phosphorylation

To perform additional Immunoprecipi- Quick CBB Plus,

2D Electrophoresis p.18 ②
separation tation sample Silver Stain MS Kit, etc.

Column Chromato-
Lysate ImmunoStar Series p.19 ③
To search for kinase or graphy + WB
inhibitors effective against Recombinant kinase
the target protein Purified protein + CBB staining, p.18 ①
Silver staining, etc.

Product Name Pkg. Size Wako Cat. No.（Nard Product #） Note

Phos-tag™ Acrylamide 0.3 mL

304-93526（AAL-107S1） Prepared aqueous solution
5 mM Aqueous Solution （0.9 mg）

2 mg 300-93523（AAL-107M）
Phos-tag™ Acrylamide Prepare with methanol or water
10 mg 304-93521（AAL-107）

■ Important differences compared to conventional SDS-PAGE and cautions

There are some important differences between Phos-tag™ SDS-PAGE and conventional SDS-PAGE that should be
considered.

◆ Prior sample preparation is required.

Due to Phos-tag™ SDS-PAGE being easily influenced by EDTA, prior sample processing (such as TCA precipita-
tion) is highly recommended ⇒ Details on page ＃14.
◆ Migration velocity is slower
Even with non-phosphorylated proteins, the migration velocity in Phos-tag™ SDS-PAGE is lower than in
conventional SDS-PAGE. ⇒ Details on page ＃17.

◆ Unable to perform molecular weight estimations using molecular weight markers.

In Phos-tag™ SDS-PAGE, molecular weight estimations using molecular weight markers is not possible. Please
simply use the markers as a guide of WB transfer efficiency. ⇒ Details on page ＃23. Additionally, pre-stained
markers can cause band distortions. It is recommended to use a recombinant or dephosphorylated sample of
the target protein as a marker. ⇒ Details on page ＃14.

◆ EDTA treatment is required for WB transfer.

In order to increase the transfer efficiency of Phos-tag™ SDS-PAGE to WB, EDTA treatment is required prior to
transfer.

◆ When performing Phos-tag™ SDS-PAGE, please concurrently perform a conventional

SDS-PAGE as a control.
This is required when multiple bands are detected in Phos-tag™ SDS-PAGE, in order to determine whether the
target protein is phosphorylated or degraded.

−５−
 3．Protocol
■Two Phos-tag™ SDS-PAGEs
Depending on the type of divalent metal ion that binds to the Phos-tag™ molecule and the composition of buffers used
in gel preparation, Phos-tag™ SDS-PAGE gels can be separated into two types as shown below. Since there are various
features available, please choose accordingly to your needs. Various examples of usage are described on page #17.

p.18-20
［Ⅰ］ Mn2+-Phos-tag™ SDS-PAGE Gel 【1】Composition based on the Laemmli method ⇒
②③④⑤

NEW！ ［Ⅱ］ Zn2+-Phos-tag™ SDS-PAGE Gel 【2】Composed of a neutral buffer ⇒p.18 ①

Gel Type Advantages Disadvantages

・Protocol similar to Laemmli method ・Some proteins are in separable.

Mn2+-Phos-tag™ ・Semi-dry transfer possible even with ・Gel preparation should be just before use.
SDS-PAGE high concentrations of Phos-tag™

・High resolving capability ・Semi-dry transfer rate is poor at high

Zn2+-Phos-tag™ ・Ability to separate a wide range of concentrations of Phos-tag™
SDS-PAGE proteins and phosphorylated forms ・Bands will smear in low concentration gels
・Long storage life of gels with less than 5%.

ABL MET FYN ABL MET FYN In Zn2+-Phos-tag™ SDS-PAGE

・Resolving capabilities were improved. (ABL, MET)
・Bands unable to be separated on Mn2+-Phos-tag™
SDS-PAGE could be resolved. (FYN)
Phosphorylated Tau
［Sample of each lane］
Left : non-phosphorylated Tau
Phosphorylated Tau Right : phosphorylated Tau
(phosphorylated form by ABL, MET, FYN)
Mn2+-Phos-tag™ Zn2+-Phos-tag™
SDS-PAGE SDS-PAGE Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced
protein phosphorylation profiling.
80μM Phos-tag™ Acrylamide, 7.5 % Polyacrylamide gel E Kinoshita and E Kinoshita-Kikuta, , Jan 2011; 11(2): 319-23.
Detection : Fluorescent staining

［Ⅰ］Mn2+-Phos-tag™ SDS-PAGE Note: Always prepare the gel just before use

① Preparation of
reagents for ：The ready-to-use solution is available. Please refer to 10. Related Products.
Phos-tag™ SDS-PAGE

Acrylamide Solution Sol. A ：30 w/v% Acrylamide/Bis Mixed

Solution (30% T, 3.3% C) Wako Catalog No. 015-25635
30 w/v% Acrylamide/Bis Mixed
Acrylamide 29.0 g Solution, 29:1 (500 mL ）

, -methylene-bisacrylamide 1.0 g
⇒ Prepare the 100 mL solution by adding distilled water and filter the solution.
【Storage】Keep at 4℃ in the dark.

Tris-HCl Buffer Sol. B ：1.5 mol/L Tris/HCl Solution, pH 8.8 (4x solution for Resolving Gel)
for Resolving Gel, # Tris base
pH 8.8 (MW：121, p a= 8.2 at 20℃) 18.2 g Wako Catalog No. 192-11041
Separating Gel Buffer Soln. (x4)
# 6.0 mol/L HCl (250 mL) containing SDS.
(0.19 equivalents of Tris) 4.85 mL

⇒ Prepare the 100 mL solution by adding distilled water and filter the solution.
【Storage】Keep at 4℃ in the dark.

−6−
 3．Protocol
Sol. C ：0.50 mol/L Tris/HCl Solution, pH 6.8 (4x solution for Stacking Gel)
Tris-HCl Buffer
for Stacking Gel, # Tris base 6.06 g
pH 6.8
# 6.0 mol/L HCl Wako Catalog No.199-11051
Stacking Gel Buffer Soln. (x4),
(0.96 equivalent of Tris base) 8.0 mL containing SDS (250 mL)

# distilled water 90 mL
⇒ Adjust the pH to 6.8 using 6.0 mol/L HCl (ca. 0.1 mL), then prepare the
  100 mL solution by adding distilled water.
【Storage】Keep at 4℃

SDS Solution Sol. D ：10% (w/v) SDS Solution

Wako Catalog No. 311-90271
# SDS 10.0 g 10% SDS Soln. (100 mL)
Wako Catalog No. 313-90275
# distilled water 90 mL 10% SDS Soln. (500 mL ）
Manufactured by Nippon Gene
⇒ After stirring, prepare the 100 mL solution
  by adding distilled water
【Storage】Keep at 4℃.

Phos-tag™ AAL Soln. Sol. E ：5.0 mmol/L Phos-tag™ Solution containing 3% (v/v) methanol
Ready-to-Use Soln. ※The amount shown in parentheses are required
for 2 mg of Phos-tag™ AAL-107
is available. Please
see the page #4 # Phos-tag™ AAL-107 (MW: 595) 10 mg
（2 mg）
(Wako Cat. No. 304-
# methanol 0.10 mL（0.02 mL）
93525)
# distilled water 3.2 mL（0.64 mL）

The oily product, Phos-tag™ AAL-107 is provided

in a small plastic tube and has to be completely
dissolved in 0.1 mL methanol.
Phos-tag™ Aqueous    ↓ Phos-tag™ Acrylamide AAL-107

Soln. is also prepared. The methanol solution should be diluted

However, it takes more with 3.2 mL of distilled water by pipetting.
time for compete Note: If a trace amount of insoluble material appears as white fine powder
dissolution than that (impurity) in the solution, it can be separated by centrifuging
the methanol soln. is (2000 x g, 10 min) using 2-mL microtubes.
prepared.
【Storage】Wrap the tube with aluminum foil.
Just after adding water into
Keep the soln. in a 2-mL microtube at 4℃ in the dark. Phos-tag™ MeOH soln.

MnCl2 Soln. Sol. F：10 mmol/L MnCl2* Solution

# MnCl2(H2O)4 (MW: 198) 0.10 g

# Distilled water 50 mL
Note: Do not use other anion salts such as Mn(NO3)2 or Mn(CH3COO)2. White precipitates
(Mn(OH)2) will be formed in basic aqueous solutions and gradually oxidize and turn
brown (MnO(OH)), and the gel will be pigmented. Also, the functions of Mn2+ will be
deteriorated.

APS Solution Sol. G：10% (w/v) Ammonium Persulfate Solution

# (NH4)2S2O8 (MW: 228) 10 mg

Wako Catalog No. 019-15922
# distilled water 0.10 mL 10 w/v% Ammonium
Peroxodisulfate Soln.
(25 mL) Ready-to-use Sol. G

−7−
 Running Buffer Sol. H：Running Buffer, pH 8.3（10x soln.）
# Tris base (0.25 mol/L) 15.1 g Wako Catalog No. 184-01291
Running Buffer Soln. (x10)
# SDS 5.0 g (1 L)

# glycine (1.92 mol/L) 72.0 g

⇒ Prepare the 0.50 L soln. by adding distilled water.
Avoid to adjust the pH by adding acid or base.
【Storage】Keep at 4℃.
Wako Catalog No. 318-90323
Just before use, add 450 mL SDS-PAGE 10x Running Buffer
of distilled water to 50 mL of Soln. H. (5 L)

Sample Buffer Sol. I：Sample Buffer（3x solution）

Wako Catalog No. 191-13272
# Bromophenol Blue (BPB) 1.5 mg Sample Buffer Soln.
(2ME+)(x4) (25 mL)
# SDS 0.60 g Wako Catalog No. 196-11022
Sample Buffer Soln.
# glycerol 3.0 mL (2ME+)(x2) (25 mL)
# Sol. C: 0.50 mol/L Tris/HCl, pH 6.8 3.9 mL
# 2-mercaptoethanol 1.5 mL
⇒ Prepare the 10 mL soln. by adding distilled water.
【Storage】Keep at -20℃.
Usage of Soln. I: Please see the“④. Sample Preparation”
（page #10）
.

Fixation Solution Sol. J：Acidic Solution for Fixation of Proteins (1 L)

# acetic acid 0.10 L
# methanol 0.40 L
# distilled water 0.50 L

CBB Staining Soln. Sol. K：CBB Staining Solution (0.5 L)

Wako Catalog No. 174-00553
# Coomassie Brilliant Blue (CBB) 1.25 g Quick CBB Plus (250 mL)

# methanol 0.20 L Wako Catalog No. 178-00551

Quick CBB Plus (1 L)
# acetic acid 50 mL
# distilled water 0.25 L
⇒ Dissolve CBB in methanol and then add acetic acid and water.

Washing and Sol. L：Washing and Destaining Solution (1 L)

Destaining Soln.
# methanol 0.25 L
# acetic acid 0.10 L
# distilled water 0.65 L

② Resolving Gel Resolving Gel Solution（0.375 mol/L Tris, 0.1 mmol/L MnCl2 , 0.1% SDS)
Preparation (In case of preparation of the 10 mL solution with 12 w/v% polyacrylamide gel and
50 μmol/L Phos-tag™ Acrylamide)

# Sol. A: 30% (w/v) Acrylamide/Bis Mixed Solution 4.00 mL

# Sol. B: 1.5 mol/L Tris/HCl Solution, pH 8.8 2.50 mL
＊1) For the MnCl2 solution, # Sol. E: 5.0 mmol/L Phos-tag™ Solution 0.10 mL
two times the Phos-tag™
concentration (molar # Sol. F: 10 mmol/L MnCl2 Solution 0.10 mL *1)
ratio) is added.
# Sol. D: 10% (w/v) SDS Solution 0.10 mL
＊2) The conventional
concentration can be # TEMED (tetramethylethylenediamine) 10 μL *2)
used for TEMED and
# distilled Water 3.14 mL
Sol.G. The above added
amounts are examples. ∼Degas by stirring for 2 minutes.∼
# Sol. G: 10% (w/v) Ammonium Persulfate Solution 50 μL *2)

−8−
 3．Protocol
Attention
Conditions should be examined for the amount of Solution E to be added. Please optimize
Phos-tag™ acrylamide concentration and acrylamide concentration for each protein of
interest. Please refer to page #17 for details.

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③ Stacking Gel Stacking Gel Solution（0.125 mol/L Tris, 0.1% SDS）

Preparation （In case of preparation of 10 mL (2 mL) of 4.5% polyacrylamide gel.)
※The amount shown in parentheses are required for the 2 mL preparation.

＊2)The normally used # Sol. A: 30% (w/v) Acrylamide/Bis Mixed Solution 1.50 mL (0.30 mL)
concentration can be
used for TEMED and # Sol. C: 0.50 mol/L Tris/HCl Solution, pH 6.8 2.50 mL (0.50 mL)
Sol.G. The above
# Sol. D: 10% (w/v) SDS Solution 0.10 mL (20μL)
added amounts are
examples. # TEMED (tetramethylethylenediamine) 10μL (2μL) *2)
# distilled Water 5.84 mL (1.17 mL)
∼Degas by stirring for 2 minutes.∼
# Sol. G: 10% (w/v) Ammonium Persulfate Solution 20∼50μL（4∼10μL) *2)

※Addition of SDS may not be necessary for resolving or stacking gels. Protein bands may become broad and
tailing may occur in gels with SDS.

  case of Separation of 200∼350 kDa Phosphorylated Proteins〉

〈In
②’
Preparation of
Resolving Gel By strengthening gels with 0.5% agarose, low concentration polyacrylamide gel
with low at 3∼5% can be prepared.
concentration
containing Resolving Gel Solution（0.375 mol/L Tris, 0.1 mmol/L MnCl2, 0.1% SDS)
agarose
（In case of preparation of 10 mL of 20 μmol/L Phos-tag ™ Acrylamide containing
3.0% Polyacrylamide gel and 0.5% Agarose）
# Sol. A: 30% (w/v) Acrylamide/Bis Mixed Solution 1.00 mL
# Sol. B: 1.5 mol/L Tris/HCl Solution, pH 8.8 2.50 mL
# Sol. E: 5.0 mmol/L Phos-tag ™ Solution 0.04 mL
＊1）
# Sol. F: 10 mmol/L MnCl2 Solution 0.04 mL
# Sol. D: 10% (w/v) SDS Solution 0.10 mL
＊2）
# TEMED (tetramethylethylenediamine) 10 μL
# distilled water 2.93 mL
＊3)Add the agarose after # 1.5% (w/v) agarose* * 3) 4)
3.33 mL
distilled water has been
＊2）
added and thoroughly # Sol. G: 10% (w/v) Ammonium Persulfate Solution 50 μL
dissolved in a microwave
oven and it is still hot.

−9−
＊4)If necessary, preheat Pour the agarose directly onto the gel preparation table before it hardens.
the pipette tip and gel
preparation table
to 40 - 45℃. Agarose H (High Strength type)
1 g (Wako Catalog No. 315-01203)
10g (Wako Catalog No. 319-01201)
25g (Wako Catalog No. 317-01202)

 
③’
Preparation of Stacking Gel Solution（0.125 mol/L Tris, 0.1% SDS）
Stacking Gel （In case of preparation of 10 mL (or 2 mL) of 3.0 (w/v)% polyacrylamide
with low
concenration containing 0.5%(w/v) agarose.)
containing
agarose # Sol. A: 30% (w/v) Acrylamide/Bis Mixed Solution 1.00 mL (0.20 mL)
# Sol. C: 0.50 mol/L Tris/HCl Solution, pH 6.8 2.50 mL (0.50 mL)
# Sol. D: 10% (w/v) SDS Solution 0.10 mL (20μL)
# TEMED (tetramethylethylenediamine) 10 μL (2μL) *2)
# distilled Water 3.01 mL (602μL)
# 1.5% (w/v) agarose* * 3) 4) 3.33 mL (666μL)
# Sol. G: 10% (w/v) Ammonium Persulfate Solution 50 μL (10μL) *2)

Pour the agarose directly onto the gel preparation table before it hardens.

④ Sample 1) Mix sample with 3 μL of Solution I (Sample Buffer) and add an appropriate amount
Preparation of distilled water to make 9 μL solution in a microcentrifuge tube.
2) Heat at 95℃ for 5 minutes, then, allow the solution to cool to room temperature.
3) Load the sample solution (eg: 1.5 μL/well) using a micropipette.
※ In case of β-casein, load 5∼10 μg /well to obtain clear bands.
  ※ It is highly recommended that impurities be removed using TCA precipitation or
dialysis, etc.

⑤ Electrophoresis 1) Assemble the electrophoresis equipments and fill the electrode chambers
with Solution H (Running Buffer).
2) Gently remove the comb from the stacking gel and load the sample into each
well using a micropipette.
3) Attach the leads to the power supply. Run the gel under a constant current
condition (25∼30 mA/gel) until the BPB reaches the bottom of the resolving gel.
(※In case of two gels, run the gels at 50∼60 mA.)

※When performing Western blotting or mass spectrometry, please refer to the page #13
below after electrophoresis.

⑥ CBB Staining 1) Just after electrophoresis, the gel is soaked in 50 mL of the Sol. J (Acidic Solution
・Destaining for Fixation of Proteins) for ca. 10 min. with gentle agitation.
2) Stain the gel by soaking in 50 mL of the Sol. K (CBB Staining Solution) for ca. 2 hours
with gentle agitation.
3) Wash the gel in 50 mL of the Sol. L (Washing and Destaining Solution) 3 times to
remove excess stain until the background is sufficiently clear.
4) Take a picture of the gel.

−10−
 3．Protocol
［Ⅱ］Zn2+-Phos-tag™ SDS-PAGE ※Gels are usable up to three months after casting. NEW！

Though the Mn2+-Phos-tag™ SDS-PAGE adopts the typical Laemmli SDS-PAGE method and is a simple procedure,
there have been cases where separation of phosphorylated and non-phosphorylated forms was not possible,
depending on the protein. In contrast, Zn2+-Phos-tag™ SDS-PAGE using the neutral Bis-Tris gel SDS-PAGE system
dramatically improves resolution, since Phos-tag™ shows the highest phosphoric acid group-capturing ability at
neutral pH.

① Preparation of Ready-to-use reagents are available. Refer to page #29∼.

reagents

Acrylamide Solution Sol. A : 30% (w/v) Acrylamide/Bis Mixed Solution (30% T, 3.3% C)
SDS Solution Sol. D : 10% (w/v) SDS Solution Please refer to[Ⅰ]
Phos-tag ™ Mn2+-Phos-tag™
Sol. E : 5.0 mmol/L Phos-tag™ Solution containing 3% (v/v) methanol  SDS-PAGE.
Acrylamide Solution
APS Solution Sol. G : 10% (w/v) Ammonium Persulfate Solution

ZnCl2 Solution Sol. M : 10 mmol/L ZnCl2 Solution Note:Please prepare it just before use.

10 mmol/L Zn(NO3)2 ZnCl2 (MW: 136, 98+%) 0. 70 g

is also usable.
distilled water 500 mL

※As ZnCl 2 is deliquescent, please use it fresh. If impurities such as ZnO can
be seen, please remove it using filtration.

Bis-Tris/HCl Solution Sol. N：1.4 mol/L Bis-Tris/HCl Solution, pH 6.8 (4x solution for resolving gel)
Bis-Tris base (MW: 209, p.a = 6.5 at 20℃) 29.9 g
6.0 mol/L HCl (0.42 equivalent of Bis-Tris) 10 mL
Prepare the 100mL solution by adding distilled water.[storage:keep at 4℃ in the dark.]

Sodium Bisulfite Sol. O：0.5 mol/L Sodium Bisulfite Solution

Solution NaHSO3 (FW: 106) 5.3 g
Prepare the 100mL solution by adding distilled water.[storage:keep at 4℃ in the dark.]
(Keep away from air.)
Running Buffer Sol. P：Running Buffer, pH 7.8 (5x solution)
Tris base (FW: 121, p.a = 8.2 at 20℃, 0.50 mol/L) 30.3 g
MOPS (FW: 209, p.a = 7.2 at 20℃, 0.50 mol/L) 52.3 g
Sol. D：10% (w/v) SDS Solution (0.5% (w/v)) 25 mL
Prepare the 500mL solution by adding distilled water. Do not adjust the pH.
[storage:keep at 4℃ in the dark.]

Running Buffer Note:Please prepare just before use.

Sol. P：Running Buffer, pH 7.8 (5x solution) 100 mL
Sol. O：0.5 mol/L Sodium Bisulfite Solution 5 mL
Prepare the 500mL solution by adding distilled water.

Resolving Gel Solution

（In case of preparation of 10mL gel with 12% acrylamide gel, 50μmol/L Phos-tag™
Acrylamide and 100μmol/L ZnCl2）
Sol. A：30% (w/v) Acrylamide Solution 4.00 mL

*1) Add ZnCl2 with double

Sol. N：1.4 mol/L Bis-Tris/HCl Solution, pH 6.8 2.50 mL
volume (molar ratio) of Sol. E：5.0 mmol/L Phos-tag™ Solution 0.10 mL
Phos-tag™ Acrylamide.
Sol. M：10 mmol/L ZnCl 2 Solution 0.10 mL＊1）
*2) The normally used
concentration can be TEMED
（tetramethylethylenediamine） 10 μL＊2）
used for TEMED and distilled water 3.24 mL
Sol.G. The above added
amounts are examples. Degas by stirring for 2minutes.
Sol. G：10% (w/v) Ammonium Persulfate Solution 50 μL＊2）

Conditions should be examined for the amount of Solution E to be added. Please

Attention optimize Phos-tag™ acrylamide concentration and acrylamide concentration for each
protein of interest. Please refer to page #17 for details.

−11−
  
Stacking Gel Solution
（In case of preparation of 10mL(2mL) of 4.5% acrylamide gel）
※The amount shown in parentheses are required for the 2mL preparation.

Sol. A：30% (w/v) Acrylamide/Bis Mixed Solution 1.50 mL（0.30 mL）

Sol. N：1.4 mol/L Bis-Tris/HCl Solution, pH 6.8 2.50 mL（0.50 mL）
TEMED（tetramethylethylenediamine） 10 μL（2 μL）＊2）
distilled water 5.94 mL（1.19 mL）
Degas by stirring for 2 minutes.
Sol. G：10% (w/v) Ammonium Persulfate Solution ＊2）
50 μL（10 μL）
※SDS addition is not necessary in resolving or stacking gels.

Please refer to【1】for sample preparation, electrophoresis, Coomassie Brilliant Blue staining and destaining. Depending
on the protein, band“smiling”may be observed. One of the reasons is that the protein's metal ligand (such as a thiol
  group) causes Zn2+ dissociation from the Phos-tag™ molecule. Adding ZnCl2 to a final concentration of 1mM in the
sample buffer will reduce smiling and improve resolution.

＜Separation of large phosphorylated proteins bigger than 200kDa＞

In conventional Zn2+-Phos-tag™ SDS-PAGE, Bis-Tris is used in the resolving and stacking gels. Bis-Tris acts as a radical
quencher since its structure is similar to TEMED. As a result, the bands smear due to a lost in sieving effect in low
concentration polyacrylamide gels (less than 5%). If performing Zn2+-Phos-tag™ SDS-PAGE using 3% or 4% low concen-
tration polyacrylamide, please prepare gels with Tris-AcOH in place of Bis-Tris. Please refer to【1】-③ as well. Recom-
mended for large proteins above 200kDa.

Composition Tris-AcOH gel Bis-Tris gel

・50mM Tris ・0.1%(w/v) SDS ・100mM Tris ・0.1%(w/v) SDS

Running buffer
・50mM Tricine ・5.0mM Sodium Bisulfite ・100mM MOPS ・5.0mM Sodium Bisulfite

Resolving / Stacking buffer ・200mM Tris-AcOH (pH 7.0) ・357mM Bis-Tris-HCl (pH 6.8)
Sample buffer ・Conventional Laemmli System ・Conventional Laemmli System

Gel concentration ・3∼4 % Polyacrylamide + 0.5 % Agarose ・≧ 5 % Polyacrylamide

−12−
 3．Protocol
■ Post-Phos-tag™ SDS-PAGE analysis
◆ Western Blotting
When transferring phosphorylated forms from Phos-tag™ SDS-PAGE gel to the membrane, transferring
NOTE
rate tends to decrease considerably. In order to improve the transfer rate, it is necessary to remove Mn2+
and Zn2+ using EDTA. Please carry out the following depending on the type of transfer apparatus used.

① Using the semi-dry method

(1) After electrophoresis, please immerse the gel in transfer buffer containing 10 mmol/L EDTA and shake gently.
(1 to 3 times for 10 min each)
※Here is an example of EDTA concentration, processing time, frequency of EDTA-containing buffer changing.
Please optimize conditions as necessary.
※Please change the EDTA-containing buffer depending on gel thickness. (Example: 1.5 mm thickness: 2 times for
20 min)
(2) Next, please immerse the gel in transfer buffer without EDTA.

Transfer proteins to PVDF membrane

Protein for 10min. × 1∼3 times
for elimination of metal ions for 10min. × 1 time

After electrophoresis Gentle agitation with Transfer buffer Gentle agitation with Transfer Gel eliminated metal ions
(containing metal ion) containing 1-10 mM EDTA. buffer without EDTA.

※Please optimize blotting conditions such as time and temperature for the target protein.
Attention When using highly concentrated Zn2+-Phos-tag™ gels (example: 100 μM Phos-tag™), sufficient transfer rate may not be attained
even after EDTA treatment. If such is the case, please use the tank method.

② Using the tank method (wet)

Please use transfer buffer containing 0.1% SDS. When using the tank method, Wako Cat. No. 019-25111
the EDTA treatment step can be omitted as long as SDS-containing transfer AquaBlot™ 10 × Tris-Glycine-
buffer is used. Pay careful attention as proteins may fall off the membrane. SDS Transfer Buffer (1L)
Please perform the procedures using SDS at the optimal concentration of 0.05%
to 0.2%

◆ Quantification analysis Wako Cat. No.

Please perform silver staining and Coomassie Brilliant Blue staining after elec- 299-58901
trophoresis, and then follow the usual in-gel digestion protocol to prepare Silver Stain MS kit
samples. EDTA treatment procedures are not necessary. (20 tests)

−13−
 4．Trouble Shooting
■ Introduction ‒ Key to success is "preparation of samples" ‒
The sample condition makes a significant difference. Prepare samples keeping the following points in mind:

● Contamination of influential substances: In particular, never use EDTA. Make sure that commercially available inhibitor
cocktails and buffers do not contain EDTA. Also eliminate EDTA from constituents of media.

● Sample condition: Do not use highly viscous samples such as overconfluent cell lysates (dilution does not improve
much).

● Standardization of buffer composition for samples: Standardize the composition of the buffer for each sample to be
applied to the gel. Pay particular attention when samples and markers treated with phosphatase are used. Apart from
chelating agents and surfactants, concentrations of MnCl2 and ZnCl2 have some influences.

If the preparation has a trouble, it is advised to re-prepare the sample paying attention to the above.

■ Popular trouble shootings (causes and actions taken)

<Problem> <Possible factors> <Actions taken>

(When a chelating agent is
the cause)
1 2 3 Do not use a prestained
marker and add 1 mM
1. Prestained Protein Size Marker(company A) MnCl2 or ZnCl2 to the
2. β-casein
3. β-casein
sample.

Prestained markers
containing a chelating The sample is contaminated
agent, etc. distort bands with a chelating agent such Prepare samples by
affecting neighboring as EDTA, vanadic acid, precipitation with TCA*,
sample lanes. inorganic salts, surfactants dialysis***, etc.
and so on.

Check the compositions of

buffers, inhibitor cocktails,
etc.

Boil thoroughly (e.g., 10

Distortion of bands minutes) and re-prepare the
sample.
The sample is viscous.
Use of overconfluent cells gives
viscosity. Improvement cannot
＊ The following sample buffers are be expected by dilution.
recommended for re-dissolution
after precipitation with TCA.
• 1 x sample buffer containing urea
(example of composition: ) If the color is yellow to
* Do not heat. orange after adding the
• 2 x sample buffer The sample is acidic.
sample buffer, add 1 M Tris
＊＊ To cleave DNA causing viscosity.
to neutralize (blue purple).
＊＊＊ Consider a dialysis tool for a small

amount of a sample.

Apply 1 x sample buffer of

There are blank lanes or the same amount as the
there is an inter-lane sample in the blank lane to
concentration gradient. eliminate the inter-lane
concentration gradient.

−14−
 4．Trouble Shooting
<Problem> <Possible factors> <Actions taken>

Perform the electrophoresis

under the condition of a low
current and long time.

The temperature is high

during electrophoresis.

Perform the electrophoresis

in a low-temperature room.
Smearing of bands

The mobility of the target Adjust the gel concentration

protein is too high (high so that the detection site of
mobility results in smearing). the target protein is in the
upper half of the gel.

Extend the duration of

treatment with EDTA and
increase the number of
exchanges of
EDTA-containing buffer.
MnCl2 or ZnCl2 is not
fully eliminated.*

Shake quite strongly when

Bad transfer to treating with EDTA.
the membrane

Also consider the following methods.

• Increase the electric current Decrease the concentration
(e.g., 200 mA). of acrylamide.

The gel is rigid.

Decrease the concentration

of methanol in the transfer
buffer.

The concentration of Add 0.5 to 2% SDS to the

Membrane after transfer by
the semi-dry method. Zn2+-Phos-tag™ is high EDTA-containing transfer
(e.g., 100 μM). buffer.
① ② ③ ④

【Sample】WIDE-VIEW™ Prestained protein Size Marker Ⅲ(Wako Cat. No.230-02461, etc.)

【Gel】 SuperSep™ Phos-tag™ (50μmol/L),12.5%, 13 wells (wako Cat. No. 196-16701)
＊
The transfer rate decreases considerably if EDTA treatment is not performed (by comparison of
① No EDTA treating
① with ②, ③ and ④). First of all, It is advised to wash twice with 1-10 mM EDTA each for 10
② 1mM EDTA for 10 minutes × 2 times
minutes. In particular, duration of the treatment and number of exchanges of EDTA-containing
③ 10mM EDTA for 10 minutes × 1 times
buffer are important.
④ 1mM EDTA for 10 minutes × 2 times

−15−
 <Problem> <Possible factors> <Actions taken>

The concentrations of Check and optimize the

Low resolution acrylamide and Phos-tag™ conditions. (See the page
are not optimal. #17 for details.)

Also consider the following methods.

• Increase the molar ratio of metal ion (MnCl2 or ZnCl2) to Phos-tag™ acrylamide in
Wako Cat. No. 200-17071
the gel (e.g. 1:4).
Tricine Running Buffer
• Use Tris-Tricine buffer as the running buffer. Solution（×10） （1 L）
• (In the case of Mn2＋-Phos-tag™ ＳDS-PAGE) Consider Zn2＋-Phos-tag™ ＳDS-PAGE.
• Prepare the reagents freshly.

■ Other trouble shootings

◆ Phosphorylated form is not obtained from immnunoprecipitation samples.
When a monoclonal antibody is used, a phosphorylated form may not be obtained due to overlapping of the epitope and
the phosphorylated region. For purification of the target protein by the immnunoprecipitation, it is advised to use a poly-
clonal antibody.
◆ The sample condition is unusual.
Washing with PBS in the preparation of cell lysate may affect the phosphorylation condition. Add TCA immediately after
elimination of the medium.
◆ It is difficult to judge whether the gel or the sample is problematic.
A mixture of phosphorylated α-casein and dephosphorylated α-casein is available as a positive control. Perform the
ordinary SDS-PAGE simultaneously using this product as a sample.
◆ Phos-tag™ acrylamide does not dissolve.
Warming at about 40°
C or treatment with an ultrasonic bath after addition of methanol and water makes dissolution easier.
◆ Separation and migration speed vary among lanes.
The concentration of the substance interfering with Phos-tag™ (such as EDTA, Mn2+, Zn2+) may differ considerably depend-
ing on the sample. Prepare samples taking care not to vary the concentration among them.

■ Protein Diffusion
Long-term migration with a constant current will cause decomposition and diffusion of proteins due to excessive heat.
①If you want to use a constant current for migration, try techniques such as using a low-temperature room, thoroughly cooling
the migration buffer just before use, and wrapping a cooling agent around the migration tank (but do not use ice because it
may cause electric shock).
②When a constant voltage can also be used, migrate with a constant voltage (eg: 200 V). The migration speed will slow down
but the generation of heat will be suppressed.

■ Easy breaking of the gel

The gel is softened due to the low concentration of acrylamide.
①5% or higher：Increasing the ’-methylene-bisacrylamide to acrylamide ratio (eg: 24：1) will strengthen the gel.
②Add 3∼5% of agarose to strengthen gels. Refer to“Preparing a Low-Concentration Gel Containing Agarose”in“3. Protocol”
and“Separation”in“7. FAQ.”

■ The background becomes high after staining.

Carry out staining after eliminating metal ions in the gel by EDTA treatment similarly to the Western blotting.

−16−
 4．Trouble Shooting
5．Optimization of Phos-tag™ SDS-PAGE Condition
To obtain a high quality result using Phos-tag™ SDS-PAGE, optimization of the concentration of acryl-
amide and Phos-tag™ Acrylamide is essential. Optimize the concentration of acrylamide (①) first,
followed by that of Phos-tag™ Acrylamide (②).

① Optimization of the concentration of acrylamide

First, identify the optimum concentration of acrylamide* that allows migration of the target protein to the
lowest end of the gel when conventional SDS-PAGE is used.
In Phos-tag™ SDS-PAGE, the migration speed is slower than in conventional SDS-PAGE (including non-
phosphorylated proteins) and, therefore, the concentration of acrylamide should be examined (see the below
figure). The migration speed decreases as the concentration of Phos-tag™ increases.

＊3VOBHFMFMFDUSPQIPSFTJTVOUJMUIF#1#EZF XIJDIJTDPOUBJOFEJOUIFTBNQMFCVGGFS SFBDIFTUIFCPUUPNPGUIFSFTPMWJOHHFM

5IFQPTJUJPOPG#1#EZFDBOCFEFmOFEBTBO3GWBMVFPG6OEFSUIFBCPWFNFOUJPOFESVOOJOHDPOEJUJPO BEKVTUUIF
PQUJNVNDPODFOUSBUJPOPGBDSZMBNJEF8IFOZPVSUBSHFUQSPUFJOJTPCTFSWFEBTBNJHSBUJPOCBOEBUBO3GWBMVFPGUPJO
DPOWFOUJPOBM4%41"(& UIFBDSZMBNJEFDPODFOUSBUJPOXPVMECFPQUJNBMGPS1IPTUBH™4%41"(&

【eg: 10% gel】

Phos-tag™ Indication
   of optimum concentration
SDS-PAGE SDS-PAGE
䠉
Ⓟ Ⓟ more than 60 kDa: 6% less than 60 kDa：8%
← Protein band ＜In case of high molecular weight proteins ＞
The gel strength can be increased by adding agarose to gels that
Ⓟ contain less than 4% of acrylamide. There are data of separation of
350 kDa. (Refer to“Separation of Phos-tag™ Acrylamide”of 7. FAQ)
Furthermore, the gel strength can also be enhanced by increasing the
-methylenebisacrylamide content (eg: 5% acrylamide [24:1]).
䠇

② Optimization of the concentration of Phos-tag™ Acrylamide

First of all, start with a lower concentration of Phos-tag™ acrylamide at 20 μM, raise the concentration up to 100 μM,
and select the concentration at which the difference in mobility between the phosphorylated form and nonphosphory-
lated form is large. Supplier of information:

Then, optimize the concentration of Phos-tag™ Acrylamide. Please evaluate eg: 20μM→50μM→100μM

5．Optimization of Phos-tag™ SDS-PAGE Condition

the optimum concentration in the order of lowest to highest.
 
【Cell Lysate】
In case there is a large variety of proteins in your sample, eg: cell lysates, the concentration of Phos-tag™
should be 5 to 25 μM. However, a higher concentration, eg: 100 μM, is recommended in case of a lower
concentration of the target protein, eg: non-overexpression systems.
※ The optimum condition depends on the protein. Please find the appropriate condition setting for each
target protein. （ ）

Relation between Phos-tag™ concentration and resolution/mobility

In general, a higher concentration leads to higher separation capacity. (Compare the samples of 50 μM and
100 μM of Mn2+-Phos-tag™ in the left figure.) However, the higher concentration causes low velocity.
It sometimes happens that the higher separation capacity is due to the lower Phos-tag™ concentration (Compare
the samples of 50 μM and 150 μM of ovalbumin of the right figure.)

【Phos-tag™ concentration and resolution】 Phos-tag™ concentration Mn2+-Phos-tag™

0 50 100 150 μM
Zn2+-Phos-tag™ and mobility
0 μM 10 μM
μ 50 μM

Phosphorylase b
（97.2 kDa）

BSA
（66.4 kDa）

Ovalbumin
（Phosphorylated, 45.0 kDa）

A：β-casein, B: Ovalbumin, C: Pepsin, M: Marker Carbonic Anhydrase (29.0 kDa)

+：Phosphorylated proteins
（A: 5Ⓟ, B: 2Ⓟ, C: 1Ⓟ）
：De-phosphorylated proteins
（Alkaline phosphatase treated） Trypsin Inhibitor (20.1 kDa)

Phos-tag™ conc. High → Separation Capacity High Phos-tag™ conc. High → Migration degree Low

−17−
 6．Application Data and References
■ Application Data 1
Impressions of users of Phos-tag™ Acrylamide and SuperSep Phos-tag™ precast gels are introduced herein.
'Ogawa provided comments and data of the sample, which had been assayed for kinase activity, separated by
Phos-tag™ SDS-PAGE. Kimura provided the application data to a secondary electrophoresis, and Hosokawa
provided the application data to the western blotting from Professor Tadayuki Ogawa of the University of Tokyo.
In addition, we received data applied to two-dimensional electrophoretic migration from Professor Yayoi Kimura
of Yokohama City University, as well as Western blotting applied data from Professor Sugiyama of Kochi Univer-
sity and Professor Hosokawa of RIKEN.

“ ” Tadayuki Ogawa, Graduate School of Medicine, the University of Tokyo

Phos-tag™ is a very convenient reagent that can be applied in a variety of samples and research purposes. It
allows quantitative analysis not only of in vitro assay samples but also in vivo samples in a phosphorylated
state. Phos-tag™ SDS-PAGE utilizes normal electrophoretic migration and does not require the purchase of
special equipment, so you could say it has good cost performance. Phosphorylation research that used to
require anti-phosphorylated antibodies, RI, and many other reagents will now be advanced with Phos-tag™.

Comparison between a phosphorylated protein and a non-phosphorylated

protein by Phos-tag™ SDS-PAGE
Various kinase-reacted phosphorylated protein
NC：Negative Control NC
BSA
Phosphorylated proteins
(red arrows)
Non-phosphorylated protein
(black arrow)
（NO3）
7.5％ acrylamide, 75μM Phos-tag™ acrylamide, 150μM Ｚｎ 2

In order to explore a kinase that phosphorylates about 40 kDa protein, the samples assayed for kinase activity were separated
by Phos-tag™ SDS-PAGE. A lot of information was obtained from a small amount of samples as, compared to nonphosphory-
lated samples (NC), the proportion of phosphorylation/nonphosphorylation, degree of phosphorylation and distribution of popu-
lation differed by the type of the kinase that reacted with other samples. With this information as the foothold, more detailed
analyses using mass spectrometry, etc. were performed to identify the phosphorylation site specific to each kinase.
(Reference : Ogawa T., Hirokawa N., ., 2015 Sep. 22 ; 12(11) : 1774-88)

■ Application Data 2
Application in two-dimensional electrophoretic migration: Analysis of phosphorylated isoforms of hnRNPK
hnRNP K was separated by immune precipitation from a nuclear homogenate of mouse macrophage cell line
J774.1 cells stimulated with LPS, and hnRNP K isoforms were separated using IPG strip gel (pH 4.7−5.9) in the
first dimension and Phos-tag™ SDS-PAGE in the second dimension. Isoforms and modification sites of the
various spots were then identified using a mass spectroscope.
Acidic 1st: Isoelectric point electrophoretic migration Basic hnRNP K:
2D heterogeneous nuclear ribonucleoprotein K
electrophoretic
migration p-Ser116/p-Ser284 (spots 1,2)
2nd：Phos-tag™ SDS-PAGE

,VRIRUP
Phosphorylated
p-Ser116 (spots 3,4,5,6) forms
p-Ser284 (spots 7,8)

Non-phosphorylated forms (spots 9,10,11,12)

,VRIRUP
,VRIRUP
※Each isoform originates in a splicing variant.
 Isoform 1,3：C termination: SGKFF
 Isoform 2,4：C termination: ADVEGF
,VRIRUP  Isoform 3,4：1 exon short
25 μM Phos-tag™ Acrylamide, 7.5% polyacrylamide gel

Different spots of phosphorylated states were detected at the same

isoelectric point for each isoform!（eg: spots 6 vs. 8 and spots 4 vs. 7）
Data published in :
Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity
electrophoresis. Y. Kimura, K. Nagata, N Suzuki, R. Yokoyama, Y. Yamanaka, H. Kitamura, H. Hirano, and O. Ohara,
, Nov 2010; 10(21): 3884-95.
Data provided by: Professors Y. Kimura and H. Hirano, Biological Supramolecular Systems, Graduate School of Bionano-
systems, Yokohama City University; and Professor O. Ohara, RCAI, Physics and Chemistry Research Institute.

−18−
■ Application Data 3

6．Application Data and References

Determining fraction containing kinase for phosphorylating Dnmt1
Dnmt1: DNA methyltransferase

Input
Pass
Western Wash 0.3 M NaCl 1 M NaCl

Phosphorylated
bands
Non-phosphorylated
bands

20 μM Phos-tag™ Acrylamide,
Fraction containing kinase
6% polyacrylamide gel
for phosphorylating GST-Dnmt1(1-290)

① GST-Dnmt1(1-290) bonding protein was obtained from mouse brain extract using affinity chromatography.
② Proteins were eluted through the DNA cellulose column by 0.3 M and 1 M NaCl.
③ In vitro kinase assay was performed in each fraction with GST-Dnmt1(1-290) as substrate.
④ Kinase activity in the fraction was confirmed by shift band, by Western blotting using Phos-tag™ SDS-PAGE
 （Detection：Anti mouse Dmnt1（72-86））

“ ”
Data published in :
The DNA-binding activity of mouse DNA methyltransferase 1 is regulated by phosphorylation with casein kinase
1delta/epsilon. Y. Sugiyama, N. Hatano, N. Sueyoshi, I. Suetake, S. Tajima, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike,
and I. Kameshita, May 2010; 427(3): 489-97.
Data provided by: Professor Y. Sugiyama, and Professor I. Kameshita, Department of Life Science, Faculty of Agriculture,
Kagawa University.

■ Application Data 4
Search for phosphorylation site of Cdk5-activated sub-unit p35 using Ala substitution variant
Cdk5: cyclin-dependent kinase 5
Regarding p35 known phosphorylation sites Ser8 and Thr138, 3 Ala substitution variants were produced (Ser8: S8A,
Thr138: T138A, Ser8 and Thr138 ：2A). These and wild-type p35, as well as Cdk5 or kinase-negative Cdk5, which has
no kinase activity, were discovered in the COS-7 cells. The cellular extract was detected by Western blotting using
Phos-tag™ SDS-PAGE. (Detected extract: anti-p35 antibody)

From lanes 1 (L2, L4) and 5 (M1): p35

Cdk5 kinase-negative wild-type is phosphorylated, depending on Cdk5.
From lanes 1 (L2, L4) and 3 (L2, L4):
Western With about half of p35, Thr138 is
A

A
38

38
A

P35 phosphorylated at kinase-negative

T

T
2A

S8

2A

S8
T1

T1
W

Cdk5, and Thr138 is also phosphory-

lated by kinase other than Cdk5.
M1（pSer8/pThr138） From lanes 5 (M1) and 6 (L3, L4):
M2（pSer8） Ser8 and Thr138 are main phosphory-
phosphorylated lation sites.
p35 L1（pThr138/X）
From lanes 5 (M1), 7 (L1, L2) and 8
L2（pThr138） (M2):
non-phosphorylated L3（X）
p35 M1 is the phosphorylation site for Ser8
L4（non-phosphorylated） and Thr138.
1 2 3 4 5 6 7 8 M2 is the phosphorylation site for Ser8
only.
100 μM Phos-tag™ Acrylamide, 7.5% polyacrylamide gel L1 and L2 are the phosphorylation
sites for Thr138 only.
※X in L1, L3：not yet identified
Relationship between phosphorylation site ※L4: non-phosphorylated p35
and band shift was clarified!
Data published in :
Quantitative Measurement of in Vivo Phosphorylation States of Cdk5 Activator p35 by Phos-tag™ SDS-PAGE.
T. Hosokawa, T. Saito, A. Asada, K. Fukunaga, and S. Hisanaga, , Jun 2010; 9: 1133 - 1143.
Data provided by: Professor T. Hosokawa, Memory Mechanisms Research Team, Circuit Function Mechanism Core,
Brain Science Research Center, RIKEN; and Professor S. Hisanaga, Nerve Molecular Functions Research Room, Bio-
science Studies, Institute of Science & Engineering, Tokyo Metropolitan University.

−19−
■ Application Data 5
Change in the phosphorylation level of MCL-1 under expression of wild-type/mutant Noxa
Wild-type (Wt) and mutant (3E, KR, 5A) Noxa genes were expressed in lung small cell carcinoma strain, H209 cells, and
further separated into the cytoplasm (Cytosol) fraction and HM (heavy membrane, containing mitochondria in large
quantities) fraction. MCL-1 (40 kDa) in these samples was isolated by Phos-tag™ SDS-PAGE and detected by Western
blotting using anti-MCL-1 antibody.

Western Cytosol HM Cytosol HM

Noxa
Nox − Wt 3E 5A − Wt 3E 5A − Wt KR − Wt KR
Phosphorylated
proteins band
MCL-1
Non-Phosphorylated
proteins band

25 μM Mn2+-Phos-tag™ Acrylamide, 10% Polyacrylamide gel

1 BH3 MTD 54
Wt Noxa Wt MPGKKARKNAQPSPARAPAELEVECATQLRRFGDKLNFRQKLLNLISKLFCSGT

3E E E E
mutant Noxa KR RR R R R R
5A AA A AA
MTD：mitochondrial targeting domain

It was found that the phosphorylation level of MCL-1 increased in mitochondria in H209
cells expressed with wild-type and KR mutant Noxa.

【Papers giving the data】

Noxa determines localization and stability of MCL-1 and consequently ABT-737 sensitivity in small cell lung cancer.
Wataru Nakajima, Mark A. Hicks, Nobuyuki Tanaka, Geoffrey W. Krystal, and Hisashi Harada
Cell Death and Disease（2014）5, e1052; doi:10.1038/cddis.2014.6
Data provided by : Dr. Wataru Nakajima, Institute for Advanced Medical Sciences, Nippon Medical School.

■ Application Data 6
Time-course change in phosphorylation after X-ray irradiation of p53 and protein X
Human lung cancer-derived Lu99 cells were irradiated with X-ray (5 Gy), and the cells were retrieved in a time course.
Cell extractions were prepared, and SDS-PAGE was performed using SuperSep Phos-tag™ (50 μmol/L), 10%, 13 wells.
The gel was shaken in the transfer buffer containing 10 mM EDTA, and transferred to a PVDF membrane. The mem-
brane was blocked with 2% Milk/TBS-T, and allowed to react with a primary antibody (upper row: p53, lower row: cell
cycle-related protein, protein X). A chemiluminescent reagent was used for detection.

Western

Time after X-irradiation − 1 2 4 6 8 （h）

p53

band shift
Protein X

SuperSep™ Phos-tag™（50μmol/L）
, 10%, 13 well

It was shown that the accumulation of p53 reached a peak after 4 hours and phos-
phorylation of protein X changed with time as a result of X-ray irradiation.

Data provided by : Dr. Atsushi Enomoto, Center for Disease Biology and integrative Medicine, Faculty of Medicine, the University of Tokyo.

−20−
 6．Application Data and References
■ References
Regarding Phos-tag™ reagents:
 .BUSJYBTTJTUFEMBTFSEFTPSQUJPOJPOJ[BUJPOUJNFPGnJHIUNBTTTQFDUSPNFUSZPGQIPTQIPSZMBUFEDPNQPVOETVTJOHBOPWFM
QIPTQIBUFDBQUVSFNPMFDVMF 3BQJE$PNNVOJDBUJPOTPG.BTT4QFDUSPNFUSZ   
)5BLFEB ",BXB
TBLJ .5BLBIBTIJ ":BNBEB BOE5,PJLF
 1IPTQIBUFCJOEJOHUBH"OFXUPPMUPWJTVBMJ[FQIPTQIPSZMBUFEQSPUFJOT .PMFDVMBS$FMMVMBS1SPUFPNJDT  

&,JOPTIJUB &,JOPTIJUB,JLVUB ,5BLJZBNB BOE5,PJLF
 4FQBSBUJPOBOEEFUFDUJPOPGMBSHFQIPTQIPQSPUFJOTVTJOH1IPTUBH™4%41"(& /BUVSF1SPUPDPMT   

&,JOPTIJUB &,JOPTIJUB,JLVUB BOE5,PJLF

Application using Mn2+- Phos-tag™ SDS-PAGE

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 +.BUPT ++-JQQ "#PHEBOPWB 4(VJMMPU &0LB[ .+VORVFJSB "4IFWDIFOLP BOE8
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 3FHVMBUJPOPGNJUPDIPOESJBMUSBOTQPSUBOEJOUFSNJDSPUVCVMFTQBDJOHCZUBVQIPTQIPSZMBUJPOBUUIFTJUFTIZQFSQIPTQIPSZ
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%JDLFZ
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 :VO$IFOBOE(FSBME8%PSO **
8. 1BSLJONFEJBUFENJUPQIBHZEJSFDUTQFSJOBUBMDBSEJBDNFUBCPMJDNBUVSBUJPOJONJDF 4DJFODF %FDBBE 
(VPIVB(POH .PTIJ4POH (ZPSHZ$TPSEBT %BOJFM1,FMMZ 4DPU+.BULPWJDI BOE(FSBME8%PSO **

Zn2+-Phos-tag™ SDS-PAGE
 1IPTQIPSZMBUJPOPG1IZUPDISPNF#*OIJCJUT-JHIU*OEVDFE4JHOBMJOHWJB"DDFMFSBUFE%BSL3FWFSTJPOJO"SBCJEPQTJT 1-"/5
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-PSSBJO 1ÏUFS(ZVMB ;TV[TBOOB.ÏSBJ $ISJTUJBO
 ."1,GFFECBDLFODPEFTBTXJUDIBOEUJNFSGPSUVOBCMFTUSFTTBEBQUBUJPOJOZFBTU 4DJ4JHOBM +BOSB +VTUJO(
&OHMJTI +BNFT14IFMMIBNNFS .JDIBFM.BMBIF 1BUSJDL$.D$BSUFS 5JNPUIZ$&MTUPO BOE)FOSJL(%PIMNBO
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4FQ 
 *DIJOPTF4 0HBXB5 BOE)JSPLBXB/
 .JDSPUVCVMF%FTUBCJMJ[FS,*'"6OEFSHPFT%JTUJODU4JUF4QFDJmD1IPTQIPSZMBUJPO$BTDBEFTUIBU%JGGFSFOUJBMMZ"GGFDU
/FVSPOBM.PSQIPHFOFTJT $FMM3FQPSUT 4FQ 


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 "øMBCPSTBWJOH UJNFTBWJOH BOENPSFSFMJBCMFøTUSBUFHZGPSTFQBSBUJPOPGøMPXNPMFDVMBSNBTTQIPTQIPQSPUFJOTJOø1IPTUBH
BGmOJUZFMFDUSPQIPSFTJT*OUø+$IFNø o 
ø,JOPTIJUB,JLVUB & ø,JOPTIJUB & BOEø,PJLF 5
 *OWJWPDPMMFDUJWFDFMMNJHSBUJPOSFRVJSFTBO-1"3EFQFOEFOUJODSFBTFJOUJTTVFnVJEJUZ +$FMM#JPM +VM
 4FJ,VSJZBNB &SJD5IFWFOFBV "MFYBOESF#FOFEFUUP .BEEZ1BSTPOT .BTBNJUTV5BOBLB (VJMMBVNF$IBSSBT 
"MFYBOESF,BCMB BOE3PCFSUP.BZPS
 %/"SFQMJDBUJPOBOETQJOEMFDIFDLQPJOUTDPPQFSBUFEVSJOH4QIBTFUPEFMBZNJUPTJTBOEQSFTFSWFHFOPNFJOUFHSJUZ +$FMM
#JPM +BO .BSJB..BHJFSB &MJTBCFUI(VFZEPO BOE&UJFOOF4DIXPC

■ Increasing number of articles on Phos-tag™

400 370

Phos-tag 283
300
240
by Google Scholar
183
200
130
67 81
100
18 36
6 3 7
0
04

05

06

07

08

09

10

11

12

13

14

15
20

20

20

20

20

20

20

20

20

20

20

20

−21−
 7．FAQ

■ Phos-tag™ Acrylamide
Quantification
Q. Can phosphorylated proteins be quantified?
A. They can be assayed on the basis of the band intensity by using a quantitative staining such as CBB staining.
A product such as“Quick-CBB PLUS”is recommended.
⇒ Quick-CBB PLUS (1 L: Wako Cat. #178-00551; 250 mL: 174-00553)

Separation
Q. Is there a limitation to the protein size for the application of Phos-tag™?
A. A phosphorylated protein of 350 kDa has actually been separated with 20 μM Phos-tag™, 3% acrylamide
and 0.5% agarose*.(See“[Ⅰ]-②’, ③’in“3. protocol”and“Tris-AcOHgel”on page 12 for details.)
Reference: , 9, 4098-4101 (2009), E. Kinoshita, E. Kinoshita-Kikuta, H. Uchijima, and K. Koike
* Agarose was added to strengthen the gel.

Staining
Q. Is it possible to use gel-staining techniques other than CBB?
A. Yes, the gel can also be stained by negative staining, silver staining, and fluorescent staining.

Use of Phos-tag™ Acrylamide

Q. How many gels can be made with each product? Phos-tag™ 20 μM 50 μM 100 μM
A. It depends on the concentration of Phos-tag™ used. 0.3 mL Aqueous
abt. 9 abt. 4 abt. 2
For example, about 100 plates at 20 μM, about 40 plates Soln. (0.9 mg )
at 50 μM, and about 20 plates at 100 μM can be prepared
2 mg package abt. 20 abt. 8 abt. 4
from a 10 mg-package, when gels of 1 mm-thickness,
9 cm-width, and 7.7 cm-length are made. 10 mg package abt. 100 abt. 40 abt. 20

SuperSep
Gel Strength ー 5 gels ー
Phos-tag™
Q. The gel is too soft. What can I do for this?
A. Please refer to "4. Trouble Shooting (page #14).

Stability of the prepared gel containing Phos-tag™

Q. How long can the prepared gel containing Phos-tag™ Acrylamide be stored?
A. Mn2+-Phos-tag™ SDS-PAGE gel cannot be stored. Use the gel on the day after preparation.
Zn2+-Phos-tag™ SDS-PAGE gel can be stored in a refrigerator for 3 months.
The gel deteriorates within a few days. Therefore, it should be prepared just before use.

Stability of the Phos-tag™ solution

Q. How long can the solutions in methanol and water be stored?
A. No remarkable decline in performance has been reported for 1 year by refrigeration under protection
from light.

−22−
 7．FAQ
■ Phos-tag™ Acrylamide
Preparation of the reagent
Q. Does the concentration of Phos-tag™ influence the amount of ions to be required? Fig. 1 Phos-tag™ Basic Structure
A. The molar ratio of Phos-tag™ acrylamide to Mn 2+
should be 1:2; two Mn
2+
ions
bind to one Phos-tag™ molecule (Fig. 1). O
O
P
O
Q. I have experienced clouding of Phos-tag™ when I prepared a solution O N
N M2+
as described in the protocol. Is this normal? M 2+
O -
N
N
A. Yes, it is. Clouding is attributed to methanol. N N
The solution becomes clear after standing for a while.
∼１ nm
Q. Does Phos-tag™ dissolve in water alone?
M ：Mn2+ or Zn2+
2+

A. It is soluble in water, though it takes more time compared to dissolution in water

containing methanol. If it does not dissolve completely, centrifuge the solution
and use the supernatant.

Fig. 2 Comparison of Prestained Markers

Molecular marker 1 2 3 4 5 6
Q. What prestained markers can we use?
A. Using a prestained marker with the Phos-tag™ gel usually causes distortion
of bands (Fig. 2). WIDE-VIEW™ Prestained Protein Size Marker III
(Wako Cat No. 230-02461) is less likely to cause band distortion, but does not
reflect the molecular weight. Please use the result obtained using this marker
as an index of the WB transfer efficiency. At least one blank lane is needed
between the solution containing this marker and other solutions.

Phosphorylation reaction with coexisting ATP

Q. Does ATP in a phosphorylation reaction solution affect electrophoresis? １，２ ：prestained marker (Company A) (3 μL)
A. ATP has no particular effect at a concentration of 2.0 mM. ３，４，５ ：WIDE-VIEWTM Prestained Protein Size Marker III (3 μL)
６ ：WIDE-VIEWTM Prestained Protein Size Marker III (5 μL)
The limit of use has not been investigated yet. Blank lanes：1 x sample buffer (5 μL)
SuperSep Phos-tagTM (50 μM), 12.5% (20 mA constant current)

Precast gel
Q. Can we use Phos-tag™ Acrylamide in an ordinary precast gel by adding it to sample solution?
A. No, you cannot. We have various kinds of precast Phos-tag™ gels called“SuperSep Phos-tag™ shown on
page #27.

Interpretation of multiple bands

Q. A band shift was observed in Phos-tag™ SDS-PAGE and multiple bands were detected. How can I decide
whether the observation demonstrates phosphorylation or just degradation of the target protein?
A. Perform an ordinary SDS-PAGE (not using Phos-tag™) simultaneously to confirm that the target protein has
not been degraded.

DNA Separation using Phos-tag™

Q. Is Phos-tag™ applicable to separate DNA?
A. Refer to the following articles:
・A SNP genotyping method using phosphate-affinity polyacrylamide gel electrophoresis,
, 361, 294-298 (2007), E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike
(The phosphate group at DNA-terminal is efficiently captured by Zn2+‒Phos-tag™.)
・A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel
electrophoresis, , 378, 102-104 (2008), E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike

−23−
■ Phos-tag™ Biotin（ ）
2
6
Q. What is the difference between BTL-104 and BTL-111? 1 BTL-104
+ + +
A. BTL-104 and BTL-111 have linkers with different lengths. +1 1+ MW:767
BTL-104 is recommendable as the first choice because 2

of its high solubility. BTL-111 offers high sensitivity. + 2

1 2 6
 1 BTL-111
Q. What is the sensitivity level like? 2 + + +
+1 1+ MW:1,367
A. It is at the nanogram level. Use a high-luminescence reagent such 2
as ImmunoStar LD.

Other reagents required

Q. Are there any reagents required other than the product?
A. Prepare a chemiluminescent reagent such as streptavidin-conjugated QJSURWHLQ
HRP solution and ImmunoStar Series. See "10" for ImmunoStar Series.















ћ&DVHLQ
Q. For how many tests can Phos-tag™ Biotin be used?
%7/
A. Please refer to the following as a guide.
BTL-104： 130∼1300 tests %7/

BTL-111 1 mM Aqueous Solution： 10∼100 tests

Q. Can phosphorylated proteins be assayed?
A. You can do semi-quantitative assay based on the density of bands.

Q. Is it possible to determine the number of bound phosphate groups?

A. No, it isn't.

Cell/tissue staining
Q. Is the product applicable to the staining of cells and tissues?
A. No. It is unsuitable for this application because it is removed by washing with methanol, etc.

Q. Can I strip Phos-tag™ Biotin?

A. Yes, you can. Mix it with a solution containing 62.5 mM of Tris-HCl (pH 6.8), 2% (w/v) of SDS, and 0.1 M
of 2-mercaptoethanol and shake the mixture for 15 minutes.
Then, wash the mixture with 1×TBS-T three times for 10 minutes each time.
For further details, please contact us.

Q. What kind of membrane is recommended?

A. We recommend PVDF membranes.

Q. Does the use of Phos-tag™ Biotin require blocking?

A. No, it doesn't. Blocking causes the sensitivity to drop.

■ Phos-tag™ Mass Analytical Kit（ ）

Q. For how many tests can Phos-tag™ Mass Analytical kit be used?
A. More than 1,000 tests when 5 μL is used per test.

Q. How can I know which one of Phos-tag™ MS-101L, Phos-tag™ MS-101H, and Phos-tag™ MS-101N is appropriate?
A. Phos-tag™ 101N contains naturally occurring zinc species, 101L contains 64Zn, and 101H contains 68Zn.
Please refer to the following guidance: ① For exploring the conditions: Use 101N.
② Many isotopes contained in it make the spectrum complicated. Verification of the presence of phosphate
groups: Use 101L and 101H.
These reagents contain zinc with a mass number of 64 and 68, respectively.
Measurement of a single sample with these reagents therefore results in a difference in m/e of 8.

−24−
 7．FAQ
Detection of non-phosphorylated molecules
Q. Why are the peaks of non-phosphorylated molecules not detected?
A. Because the ionization efficiency differs considerably between phosphorylated molecules and nonphosphory-
lated molecules. The sample solution using Phos-tag™ is suited to the method using a buffer of pH 6 to 8 and a
weak-acidic phenol matrix (such as THAP) and weak-basic HAMAN.
In the peptide analysis in the general positive mode, on the other hand, acidic sample solutions and acidic
matrix are used.
Therefore, the ionization efficiency of the phosphorylated molecule-Phos-tag™ complex increases dramatically
whereas that of the non-phosphorylated molecules becomes extremely low.

Q. I would like to measure a sample isolated by Phos-tag™ SDS-PAGE. Is it necessary to remove Phos-tag™
before in-gel digestion?
A. No, it isn't. Please follow the usual procedure for in-gel digestion after SDS-PAGE.

Q. Can it be also used for ESI mass spectrometry?

A. Yes, it can. Please refer to the following publication, which reports an example of ESI-MS analysis in which
Phos-tag™ MS-101N was used as probe. A neutral solution should be used because analysis in an acidic
solution causes Phos-tag™ to be detached.
Reference: . (2008), 80, 2531-2538 (MS-101N ESI-MS)

■ Phos-tag™ Agarose（ ）

Q. Can samples purified by using Phos-tag™ Agarose be directly applied to SDS-PAGE?

A. No, they can't. The elution buffer recommended in the protocol contains a high concentration of salt and
may cause the bands to be distorted. Please use the SDS-PAGE sample buffer as elution buffer.

Q. Is Phos-tag™ Agarose reusable?

A. We do not recommend it.

Q.Does Phos-tag™ Agarose have any advantages over IMAC?

A. Phos-tag™ Agarose allows experimental processes under physiological conditions (pH 7.5), and since it
does not use reductants or surfactants, it can refine phosphorylated proteins in their native shape.
Also, the purified proteins can be used in processes such as mass spectrometry and Western blotting.
Purification of His-tag proteins
Q. Is the product applicable to purification of His-tag phosphorylated proteins?
A. As His-tag exhibits weak affinity to Zn2+, use a tag of other types such as GST, if possible.
It is assumed that Zn2+ shows a higher affinity to Phos-tag™ than to His-tag since isolation of His-tag protein by
Zn2+ -Phos-tag™ SDS-PAGE has been reported although no result of purification of His-tag protein with Phos-tag™
Agarose has been confirmed.

Q. What reagents are suitable and unsuitable for use in the sample preparation?
A. Please refer to the table below.

Category Reagent Suitability Allowable concentrations

Reducing agents DTT ○ ≤ 0.1 M
Denaturing agents Urea ○ Using it at 8M has no negative effect.

Surfactants SDS ○ Using it at ≥ 0.5% affects the binding process.

(anionic) Sodium deoxycholate ○ Using it at ≥ 0.25% affects the binding process.

Surfactants Nonidet P40 ○ ≤1 %

(nonionic) Tween 20 ○ ≤1 %

Surfactants
CHAPS ○ ≤ 0.2 %
(amphoteric)

β-Glycerophosphate × Do not use it.

Phosphate derivatives
Pyrophosphate × Do not use it.
Chelating agents EDTA × Do not use it.

−25−
8．SuperSep Phos-tag™
SuperSep Phos-tag™ is a precast gel, which can be immediately used after opening the package. It contains
zinc as a supplemental metal. It has excellent storage stability by its neutral gel buffer and ZnCL2. Sharp
bands can be obtained.

Features ● Ready-to-use
● Safety due to precast gel
● Long-term stability (Stable for 9 months)
● Almost the same basic mechanism as normal SDS-PAGE
● High reproducibility
SuperSep Phos-tagTM

Plate Size 100 x 100 x 3 (mm) ZnCl2 conc. 100 μmol/L

Gel Size 90 x 85 x 1 (mm) Well volume 30 μL 25 μL
Well number 13 17 Storage Condition Keep at 2∼10℃
Phos-tag™ conc. 50 μmol/L

・Use of a normal prestained marker will distort the bands. Use of WIDE-VIEW™ Prestained Protein Size Marker III
(Wako Cat. No. 230-02461) is recommended. Please refer to Phos-tag™ Acrylamide - Molecular Marker
ATTENTION in 7. FAQ. in the page #23.
・Before using this product, check a sample for migration pattern problems with SuperSep Ace or other normal SDS-PAGE.
・When performing Western blotting, execute an EDTA process before transfer. For details, refer to 4. Troubleshooting.

■ Application ∼ Dephosphorylation over time of β-casein ∼ Refer to the page #20 on WB data.

SuperSep Ace, SuperSep Phos-tag™, ［ Buffer］

12.5%, 13 wells 12.5%, 13 wells Tris-Glycine-SDS electrophoresis buffer
［ Sample］
M: WIDE-VIEW™ Prestained
Protein Size Marker III (Wako Cat.
No. 230-02461)
1 : 0 min. β-casein (with AP treatment)
2 : 15 min. β-casein (with AP treatment)
㻌㻹㻌㻌 㻝㻌㻌㻌㻞㻌㻌㻌㻟㻌㻌㻌㻠㻌㻌㻌㻡 㻌㻹㻌㻌 㻌㻝㻌㻌㻌㻌㻞㻌㻌㻌㻌㻟㻌㻌㻌㻌㻠㻌㻌㻌㻡 3 : 30 min. β-casein (with AP treatment)
4 : 45 min. β-casein (with AP treatment)
5 : 60 min. β-casein (with AP treatment)
［ Condition］
Constant current 35 mA for 60 min.
Phosphorylated ［ Staining］
Phosphorylated
β-casein Quick-CBB Staining
β-casein
（Wako Cat. No. 299-50101）
＋
dephosphorylated Dephosphorylated ［ Destaining］
β-casein Deionized water with microwave
β-casein
β-casein was dephosphorylated over time.
Dephosphorylated β-casein can be separated
from β-casein with SuperSep Phos-tag™.

■ Product List
（1）for Bio-Rad's Electrophoresis Tank
Product Name Pkg. Size Wako Cat. No. Electrophoresis Tank
SuperSep Phos-tag™ (50 μmol/L), 7.5%, 17 wells 83×100×3.9 mm 198-17981 Mini-PROTEAN® Tetra Cell
5 gels
SuperSep Phos-tag™ (50 μmol/L), 12.5%, 17 wells 83×100×3.9 mm 195-17991 (Bio-Rad Laboratories, Inc.)
Mini-PROTEAN is a registered trademark of Bio-Rad Laboratories, Inc.
（2）for Life Technologies' Electrophoresis Tank
Product Name Pkg. Size Wako Cat. No. Electrophoresis Tank
SuperSep Phos-tag™ (50 μmol/L), 7.5%, 17 wells 100×100×6.6 mm 192-18001 XCell SureLock™ Mini-Cell
5 gels
SuperSep Phos-tag™ (50 μmol/L), 12.5%, 17 wells 100×100×6.6 mm 199-18011 (Life Technologies, Inc)
XCell SureLock is a registered trademark of Life Technologies, Inc.
（3）for Wako s Electrophoresis Tank
Product Name Wako Cat. No. Pkg. Size Electrophoresis Tank
SuperSep Phos-tag™（50μmol/L）, 6%, 13 wells 192-17401
SuperSep Phos-tag™（50μmol/L）, 6%, 17 wells 199-17391
SuperSep Phos-tag™（50μmol/L） , 7.5%, 13 wells 195-17371
SuperSep Phos-tag™（50μmol/L） , 7.5%, 17 wells 192-17381 EasySeparator
SuperSep Phos-tag™（50μmol/L）, 10%, 13 wells 193-16711
5 gels
SuperSep Phos-tag™（50μmol/L）, 10%, 17 wells 190-16721
SuperSep Phos-tag™（50 μmol/L） , 12.5%, 13 wells 195-16391
SuperSep Phos-tag™（50 μmol/L）, 12.5%, 17 wells 193-16571
SuperSep Phos-tag™（50 μmol/L）, 15%, 13 wells 193-16691
SuperSep Phos-tag™（50 μmol/L） , 15%, 17 wells 196-16701
These SuperSep Phos-tag™ precast gels are designed for the EasySeparator' tank.
EasySeparator™ 058-07681 1 unit ─

−26−
 8．SuperSep Phos-tag™
9．Phos-tag™ Series

Product Name Pkg. Size Wako Cat. No. Maker s code No. Applications

Phos-tag™ Biotin BTL-111 BTL-111 offers higher sensitivity than

0.1 mL 308-97201 BTL-111S1
1mM Aqueous Solution Ref.1) BTL-104.
Specific detection without any
anti-phosphorylated antibodies on Western
Phos-tag™ Biotin BTL-104 10 mg 301-93531 BTL-104
blot. Detection is possible regardless of the
type of phosphorylated amino acid.

Phos-tag™ Mass Analytical Kit 1 Kit 305-93551 MS-101KIT Analysis by MALDI-TOF/Mass

0.5 mL 302-93561 AG-501

Phos-tag™ Agarose Enrichment, separation and purification of
3 mL 308-93563 AG-503 phorphorylated proteins using column
chromatography
Phos-tag™ Tip 8tips 387-07321 AG2-103

■ Phos-tag™ Biotin ― Detection of phosphoprotein by Western blotting ―

This biotin-bound Phos-tag™ is used for the detection of phosphoprotein by Western blotting.

Features ● Recommended when anti-phosphorylated antibodies are not available regardless of the type
of phosphorylation.
● No special reagent or device is necessary.

It can be conveniently used even if target

BTL-104 
anti-phosphorylated Thr/Ser antibody is not available !
M.W. 767
※ BTL-104, and BTL-111 have linkers with different lengths. BTL-111 offers
higher sensitivity.
BTL-111 
M.W. 1,367
Schematic of western blotting using Phos-tag™ Biotin
Application
Luminescent substrate ∼Detection of β-casein, Ovalbumin∼
+53 β-casein

9．Phos-tag™ Series
Phos-tag™ Biotin
6WUHSWDYLGLQ
1
2 BTL-111
+1
+
1 2 %LRWLQ
1
=Q 
BTL-104
1 2
2
=Q 
AP treated
1 2 3 2
2 (200 ng)
(PVDF membrane)
BTL-111
Phosphorylated protein Non-phosphorylated protein
Phosphorylated proteins on PVDF membrane can be detected similarly BTL-104
to the anti-phosphorylation antibody in Western blotting.
200 0.1
Protein
（ng）

■ Phos-tag™ Mass Analytical Kit

−Detection Sensitivity of MALDI-TOF/Mass is improved −
Before use, Phos-tag™ Mass Analytical Kit is mixed with samples for MALDI-TOF/Mass analysis. Phosphorylated
molecule-Phos-tag™ complex is detected in a positive mode, and phosphorylated molecules usually difficult to detect
can be detected with improved sensitivity.Three types of reagents contain different types of zinc.

Application
Feature ● Detection sensitivity of phosphorylated molecule is
improved. ∼Detection of Phos-tag™-phosphorylated
● Non-phosphorylated molecules are not detected. LPA complex∼
● Applicable to phosphorylated molecules other than 25000

phosphorylated peptides. 20000

【Kit Contents】 15000

Counts

・Phos-tag™ MS-101L ……………… 5 mg

 （［C27H29N6O64Zn2］3+ MW：581.4） 10000
The detection sensitivity
・Phos-tag™ MS-101H ……………… 5 mg of phosphorylated LPA
5000 is improved.
 （［C27H29N6O68Zn2］3+ MW：589.4）

・Phos-tag™ MS-101N ………………10 mg 0

 （［C27H29N6OZn2］3+  MW：584.3） 400 600 800 1000 1200 1400
Mass
（m/z）
The detection sensitivity of phosphorylated LPA is improved.
LPA - :1-oleoyl-L-α-lyzophosphatidate
2
（M.W. : 434.2）

−27−
■ Phos-tag™ Agarose
− Purification of phosphorylated proteins by affinity chromatography −
Fill Phos-tag™ Agarose in a column for use. It can be used for
separation, purification and concentration of phosphorylated
proteins. Because it is free of surfactants and reducing agents,
phosphorylated proteins can be obtained in a condition similar to
.

Features ● Phosphorylated proteins can be purified

Crosslinked
within 1 hour. agarose gel
beads
● The proteins can be trapped
in physiological condition (pH 7.5).
Phos-tag™ Agarose
● Purified without reducing agent and surfactant.

Application
∼Purification of phosphorylated proteins in A431 lysate∼
M1 2 3 M 1 2 3
M：Molecular Marker
Lane 1：Non adsorbed fraction
Adsorb Wash Elute Lane 2：Adsorbed fraction
Lane 3：Column rinsing fraction
（Left）Fluorescence staining
（right）Western blotting with
anti-phosphorylated Tyr

Phosphorylated protein
Lysate was applied to a
Non-Phosphorylated protein column filled with
Phos-tag™ Agarose Phos-tag™ Agarose.
Phosphorylated proteins
were concentrated in
the adsorbed fraction.

■ Phos-tag™ Tip
− Ready-to-use Phos-tag™ tip for the concentration of phosphorylated peptides −

Features ● The operation time is less than

Crosslinked
30 minutes. agarose gel
● High recovery rate beads

● No expensive device required

Phos-tag™ Agarose
Phos-tag™ Tip

Application
∼Isolation of substances digested with 6 nmol β-casein trypsin∼
Binding washing elusion P1 : FQpSEEQQQTERELQDK
P2 : RELEELDVPGEIVEpSLpSpSpSEESITR

Detection
sample
（HPLC）
p （215nm）

Phos-tag™ gel

Flowthrough Fraction
Attach the tip to the
Adsorption

syringe (accessory to
the product) for use.
Elution Fraction

Elution Time（0-22 min）

−28−
 9．Phos-tag™ Series
Related Products（Electrophoresis/Western Blotting）
10．
■ Reagents for Phos-tag™ SDS-PAGE gel preparation
Product Name Pkg. Size Wako Cat. No. Use Application

30 w/v% Acrylamide / Bis Mixed solution（29:1） 500 mL 015-25635 Ready-to-use Solution A . 30%T, 3.3%C

1g 315-01203 High-strength Agarose has high strength even

in a low-agarose environment and is suitable for
Agarose H (High-strength type) 10 g 319-01201 electrophoretic migration of large nucleic acid
fragments. It can be used in a concentration range
25 g 317-01202 of 0.2 - 1% and a separation range of 1 ‒ 200 kbp.

100 mL 311-90271
10% SDS Solution Ready-to-Use Solution D
500 mL 313-90275

Manganese(II) Chloride Tetrahydrate, 25 g 134-15302 for Molecular Biology

99.0+ % (Titration) Please use for preparation of Solution F
100 g 136-15301

Zinc Chloride 25 g 268-01902 for Molecular Biology. Please use for preparation of Solution M

Bis-Tris 100 g 345-04741 Please use for preparation of Solution N

25 g 196-01372
Sodium Hydrogensulfite〈JIS Special Grade〉
100 g 198-01371 Please use for preparation of Solution O

Sodium Hydrogensulfite〈For Molecular Biology〉 100 g 190-16461

100 g 345-01804
MOPS
250 g 341-01801
Please use for preparation of Solution P
100 g 341-08241
MOPS〈For Molecular Biology〉
500 g 343-08245

Ready-to-Use mixed solution of Sol. B and Sol. D

Separating Gel Buffer Solution (x4) 250 mL 192-11041
for preparation of Resolving Gel. Contains SDS.

Ready-to-Use mixed solution of Sol.C and Sol. D

Stacking Gel Buffer Solution (x4) 250 mL 199-11051
for preparation of Stacking Gel. Contains SDS.

-Tetramethylethylenediamine (TEMED) 25 mL 205-06313 for Electrophoresis

10．Related Products（Electrophoresis/Western Blotting）

10 w/v% Ammonium Peroxodisulfate Solution
25 mL 019-15922 Ready-to-Use Solution G
(Ammonium Persulfate Solution)

■ Premixed Buffers
Product Name Pkg. Size Wako Cat. No. Use Application

Running Buffer Solution (x10) 1L 184-01291 Ready-to-Use concentrated Solution H

1L 312-90321
SDS-PAGE 10x Running Buffer Ready-to-Use concentrated Solution H
5L 318-90323

SDS-PAGE Buffer, pH 8.5 5L 192-16801 Ready-to-Use Solution H , 1 x buffer.

Tricine Running Buffer Solution (x10) 1L 200-17071 Composition: 0.5 M Tris / 0.5 M Tricine / 1% SDS

Sample Buffer Solution (2ME+) (x4) 25 mL 191-13272 Sample buffer for Laemmli SDS-PAGE
containing 2-mercaptoethanol
Sample Buffer Solution (2ME+) (x2) 25 mL 196-11022

Sample Buffer Solution

25 mL 199-16132
with 3-Mercapto-1,2-propanediol (x2) Laemmli Sample Buffer containing
3-mercapto-1,2-propanediol (non-hazardous
Sample Buffer Solution chemical) as substitute for 2-ME
25 mL 196-16142
with 3-Mercapto-1,2-propanediol (x2)

AquaBlot™ 10× Tris-Glycine-SDS Transfer Please use it to transfer to a membrane. Containing

1L 019-25111
Buffer 0.05% SDS

■ Enzyme for Dephosphorylation

Product Name Pkg. Size Wako Cat. No. Use Application

50 U 018-10693
Alkaline Phosphatase（for Biochemistry） Applicable to dephosphorylation of proteins
100 U 012-10691

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■ Reagents for Staining
Product Name Pkg. Size Wako Cat. No. Use Application

Ready-to-Use Sol. K.
250 mL 174-00553
Fixing and destaining procedure are not required.
Quick CBB Plus
No organic solvents are necessary.
1L 178-00551 Protein bands are stained in 10 minutes.

Quick-CBB
・Staining solution A: 1L × 1 2L 299-50101 By mixing staing solution A and B, ready-to-Use Sol. K
・Staining solution B: 1L × 1

Modified proteins by glycosilation and / or

Silver Stain MS Kit 20 tests 299-58901 phosphorylation can be detected sub-nanogram level
on the electrophoretic gel.

Silver Stain Kit for10 gels 299-13841 50 100 times more sensitive than CBB method.

This kit contains Stopper, which can be adjusted

Silver Stain II Kit for10 gels 291-50301
the staining strength.

Negative Gel Stain MS Kit 20 tests 293-57701 Applicable to mass analysis and Western blot

■ Protein Size Marker

Product Name Pkg. Size Wako Cat. No. Use Application

25 µL 236-02463
A recommendable prestained marker used
WIDE-VIEW™ Prestained Protein Size Marker III 500 µL 230-02461 with Phos-tag™ SDS-PAGE because obtained bands
are less distorted.
500 µL × 3 234-02464

■ Positive Control（for confirmation of separation capacity of Phos-tag™ SDS-PAGE）

Product Name Pkg. Size Wako Cat. No. Use Application

1 mg 038-23221 Mixture of phosphylated and dephosphorylated

α-Casein, from BovineMilk, Dephosphorylated
α-casein
10 mg 034-23223

■ Electrophoresis Apparatus ・ Precast Gels

Product Name Pkg. Size Wako Cat. No. Use Application

An electrophoresis tank for SuperSep precast

EasySeparator 1 unit 058-07681
polyacrylamide gels.

SuperSep Ace, 6%, 13 wells 10 gels 195-15171

SuperSep Ace, 7.5%, 13 wells 10 gels 198-14941

SuperSep Ace, 7.5%, 17 wells 10 gels 191-14931

SuperSep Ace, 10%, 13 wells 10 gels 195-14951 Prior to use of SuperSep Phos-tag™ PAGE,
SuperSep Ace, 10%, 17 wells 10 gels 192-14961 please use these as sample confirmation.
Expired in 9 months after manufacture
SuperSep Ace, 12.5%, 13 wells 10 gels 199-14971

SuperSep Ace, 12.5%, 17 wells 10 gels 196-14981

SuperSep Ace, 15%, 13 wells 10 gels 193-14991

SuperSep Ace, 15%, 17 wells 10 gels 190-15001

■ Reagents for Western Blotting

Product Name Pkg. Size Wako Cat. No. Use Application

200 ㎝ 2
296-69901 Highly sensitive (femto gram level) immunoblotting,
ImmunoStar LD*
・Luminescence solution A utilizing detection by enhanced chemiluminescence
1,000 ㎝ 2
292-69903
・Luminescence solution B using a unique luminol derivative L-012 as a substrate.
2,000 ㎝ 2
290-69904 Not available for sale in the US and Europe.

200 ㎝2 291-72401
Use for detection of proteins between the middle
ImmunoStar Zeta* 1,000 ㎝2 297-72403 and low femto gram levels. Has stable, long-lasting
luminescence signal.
2,000 ㎝2 295-72404

200 ㎝ 2
295-75101

ImmunoStar Basic* 2,000 ㎝ 2

291-75103 A cost-effective, stable and long-lasting product.

5,000 ㎝ 2
299-75104

2 assays 294-68601
Ready-to-Use Immunoreaction Enhancer
Immuno Enhancer 10 assays 290-68603
for Western blotting and ELISA
40 assays 298-68604
＊ : Not available for sales in the US and Europe.

−30−
10．Related Products（Electrophoresis/Western Blotting）

−31−
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