Electrophoresis

•Electrophoresis is the motion of dispersed charged

particles relative to a fluid under the influence of a spatially
uniform
if electric
l t i field
fi ld - it is
i a primary
i t h i
technique t separate
to t andd
analyze biological molecules where Charged particles migrate
•It takes advantageg of the fact that p
proteins and nucleic acids are
generally charged and therefore move in an electric field towards
the positive or negative electrode depending on their charge
•As an analytical tool,
tool electrophoresis is simple,
simple rapid and highly
sensitive
•It can be used analytically to study the properties of a single
charged species or mixtures of molecules. It can also be used
preparatively as a separating technique
•Their rate of migration through the electrical field,
field depends on the
strength of the field, on the net charge, size, and shape of the
molecules, and also on the ionic strength, viscosity, and
temperature of the medium in which the molecules are moving
 Electrophoresis

This expression
p was used to measure the electron charge g at
the turn of the last century, but biological molecules have
complex electrostatic properties, because they are surrounded
b counterions
by t i (
(e.g. mono and
d divalent
di l t ions
i f nucleic
for l i acids)
id )
that shields the electrostatic field in a complex way. As it
moves the molecule also drags its ionic atmosphere along
moves,
with it, thereby affecting the frictional coefficient that
depends in complex ways y on the shape and charge g of the
molecule and on the nature of the electrophoretic medium.
For these reasons, it is very difficult to measure absolute
properties
ti off biological
bi l i l molecules
l l b
by electrophoresis.
l t h i
However, because the technique is so sensitive, it is used very
effectively to separate molecules that differ very little in
charge and/or mass
 A FEW THINGS TO KNOW ABOUT ELECTROKINETICS
The electric field exerts a force on the ions in the diffuse layer which has direction
opposite to that acting on the surface charge. This latter force is not actually
applied to the particle, but to the ions in the diffuse layer located at some distance
from the particle surface acting like viscous stress and is known as the
electrophoretic retardation force (ERF). When the electric field is applied and the
charged particle to be analyzed is at steady movement through the diffuse layer, the
total resulting force is zero :
In a fluid in the steady state, the flow equations are
qE − f f − fv ≈ 0
In case of low Re, a thin layer of fluid (<<
DL, 1/κ) and moderate electric field
strength
h E,
E we assume friction
f i i =0, 0 so that
h

f v = 6πRηv
ε r ε 0ζ
v = μ e E⇒μ e =
q
=
Ze
or μe =
6πRη 6πRη η
Where εr is the dielectric constant of the dispersion medium, ε0 is the permittivity of free
space μe is called the electrophoretic mobility.
 Electrophoresis
• Electrophoresis is usually done with gels formed in tubes, slabs, or
on a flat bed.
• In many electrophoresis units,
units the gel is mounted between two
buffer chambers containing separate electrodes, so that the only
electrical connection between the two chambers is through the gel.
Gel Units
A microfluidic system for DNA separation

From Agilent
Agilent-
Caliper

Allow to characterize DNA

Fragments with excellent
Resolution, and in a small
time
Interrelation of Resistance, Voltage, Current and Power
• Two basic electrical equations are important in
electrophoresis
– The first is Ohm's Law, I = E/R
– The second is P = EI
– This
Thi can also
l be
b expressed d as P = I2R
• In electrophoresis, one electrical parameter, either
current voltage
current, voltage, or power
power, is always held constant
• Under constant current conditions (velocity is directly
proportional to current), the velocity of the molecules
is maintained, but heat is generated.
• Under constant voltageg conditions,, the velocity
y slows,,
but no additional heat is generated during the course
of the run
• Under constant power conditions, the velocity slows
but heating is kept constant
 Temperature and Electrophoresis
• Important at every stage of electrophoresis
– During Polymerization
• Exothermic Reaction
• Gel irregularities
• Pore size
– During Electrophoresis
• Denaturation of proteins
• Smile effect
• Temperature Regulation of Buffers
• Proteins are amphoteric compounds, that is, they
contain both acidic and basic residues
• Each protein has its own characteristic charge
properties depending on the number and kinds of
amino acids carrying amino or carboxyl groups
• Nucleic acids,, unlike p proteins,, are not amphoteric.
p
They remain negative at any pH used for
electrophoresis
 The Solid Support Matrix: Agarose and Polyacrylamide
• It iinhibits
hibit convection
ti and d diff
diffusion,
i which
hi h wouldld otherwise
th i iimpede
d
separation of molecules
• It allows a permanent record of results through staining after run
• It can provide additional separation through molecular sieving
• Although agarose and polyacrylamide differ greatly in their physical and
chemical structures, they both make porous gels.
• A porous gel acts as a sieve by retarding or, in some cases, by completely
obstructing the movement of macromolecules while allowing smaller
molecules to migrate freely.
• By preparing a gel with a restrictive pore size, the operator can take
advantage of molecular size differences among proteins
• Because the p pores of an agarose
g ggel are large,
g , agarose
g is used to separate
p
macromolecules such as nucleic acids, large proteins and protein
complexes
• Polyacrylamide, which makes a small pore gel, is used to separate most
proteins and small oligonucleotides.
• Both are relatively electrically neutral
 Agarose Gels
• Agarose is a highly purified uncharged polysaccharide
derived from agar
• Agarose dissolves when added to boiling liquid. It remains in
a liquid state until the temperature is lowered to about 40° C
at which point it gels
• The pore si sizee ma
may be predetermined b
by adj
adjusting
sting the
concentration of agarose in the gel
• Agarose
g ggels are fragile,
g however. They y are actuallyy
hydrocolloids, and they are held together by the formation of
weak hydrogen and hydrophobic interactions
Basic
B i di
disaccharide
h id
repeating units of
agarose,

G: 1,3-β-d-galactose

and

A: 1,4-α-l-3,6-
anhydrogalactose
 Polyacrylamide Gels
• Polyacrylamide gels are
tougher than agarose gels
• Acrylamide monomers
polymerize into long chains
that are covalently linked by
a crosslinker
• Polyacrylamide is chemically
complex, as is the production
and use of the gel
 Considerations with PAGE
• Preparing and Pouring Gels
– Determine pore size
• Adjust
j total p
percentage
g of acrylamide
y
• Vary amount of crosslinker
– Remove oxygen from mixture
– Initiate
ii polymerization
i i
• Chemical method
• Photochemical method
• Analysis of Gel
– Staining or autoradiography followed by
densitometry
– Blotting to a membrane, either by capillarity or by
electrophoresis, for nucleic acid hybridization,
autoradiography or immunodetection
 Gel electrophoresis of proteins

Unlike nucleic acids, proteins are not uniformly charged and

their net charge depends not just on the sequence of the
proteins, but also on the pH of the solution

Furthermore, like RNA molecules or topoisomers, proteins

will adopt different structures and therefore shapes, which
will determine the mobility of the protein under
electrophoretic conditions

One could use these properties to study the shape of proteins

but most often,
but, often electrophoresis of proteins is used to
determine the molecular weight of protein samples and is
therefore conducted under conditions where proteins are not
structured (high concentrations of a surfactant, SDS, that
causes proteins to denature)
 Gel electrophoresis of proteins

An additional advantage of having SDS is that at

concentrations higher than mM, most proteins will bind a
nearly constant amount of SDS per amino acid
(approximately 0.5
0 5 molecules of SDS per amino acid,
acid on
average)

Under these conditions, the charge of the SDS/protein

complex is determined primarily by the surfactant itself,
making the charge per unit weight independent of the protein
sequence and therefore the same for every protein. Under
these conditions
conditions, the mobility of SDS-treated
SDS treated proteins is
determined only by the protein molecular weight;
p y, there is a linear relationship
experimentally, p between the Log
g
of molecular weight and distance x traveled in the gel.
 SDS Gel Electrophoresis
• In SDS separations, migration is
determined not by intrinsic
electrical charge of polypeptides
but by molecular weight
• Sodium dodecylsulfate (SDS) is an
anionic detergent which
hich denatures
denat res
secondary and non–disulfide–
linked tertiaryy structures byy
wrapping around the polypeptide
backbone. In so doing, SDS
confers a net negative charge to
the polypeptide in proportion to
its length
• When
Wh treated
d with
i h SDS and d a
reducing agent, the polypeptides
become rods of negative charges
with equal “charge densities" or
charge per unit length.
 Continuous and Discontinuous Buffer Systems
• A continuous system has only a single separating gel and
uses the same buffer in the tanks and the gel
• In a discontinuous system a nonrestrictive large pore gel,
called a stacking gel, is layered on top of a separating gel
• The resolution obtainable in a discontinuous system
y is
much greater than that obtainable in a continuous one.
However, the continuous system is a little easier to set up
Coomassie Blue Staining
Silver Staining
 Gel electrophoresis of proteins

The mobility of SDS-treated proteins is determined only by

the protein molecular weight; experimentally, there is a linear
relationship between the Log of molecular weight and
di t
distance x traveled
t l d in
i the
th gel:
l

log M = a − bx

Where a and
Wh d b are constant d
determined
i db by the
h electric
l i fi
field
ld
strength and gel matrix
 Gel electrophoresis of proteins

log M = a − bx

This pproperty
p y can be used to analyze y p protein molecular
masses, although for some proteins that are highly charged
(e.g. histones) or that binds unusual amounts of SDS
(glycoproteins) or heavily modified (phosphorylated or
methylated or acetylated proteins), or intrinsically
unstructured domains this relationship is not true.
true Deviations
from this behavior can therefore be used to monitor the
modification state of a pprotein ((e.g.
g its p
phosphorylation!).
p y )
 Determining Molecular Weights of Proteins by SDS-PAGE
• Run a gel with standard proteins of
known molecular weights along with
the polypeptide to be characterized
• A linear relationship exists between
the log10 of the molecular weight of a
polypeptide and its Rf
• Rf = ratio of the distance migrated by
the molecule to that migrated by a
marker dye-front
• The Rf of the ppolypeptide
yp p to be
characterized is determined in the
same way, and the log10 of its
molecular weight is read directly
from the standard curve
 Electrophoretic Blotting
• Blotting is used to transfer proteins or nucleic acids from a slab
gel to a membrane such as nitrocellulose, nylon, DEAE, or CM
paper
• The transfer of the sample can be done by capillary or Southern
blotting for nucleic acids (Southern, 1975) or by electrophoresis
f proteins
for i or nucleici acids
i
 Gel electrophoresis of nucleic acids

The path traveled by the molecule through the porous gel is

very long, much greater than the length of the gel

The gel imposes additional frictional forces on the molecule

The macromolecules interact with charged

g ggroups
p on the ggel
network

The pore size

Th i may b be too small
ll for
f certain
i molecules
l l to
penetrate the gel matrix

All of these factors allow separation to be conducted with

exquisite
q sensitivity
y to very
y small differences in mass, charge
g
and shape
 Electrophoresis of DNA

Electrophoresis under native conditions in either agarose (a

mixture of a polysaccharide derived from algae) or
acrylamide
y can be used not onlyy to separate
p nucleic acids
based on their size but also based on their conformation

Double
D d d DNA - can be
bl stranded b separated
t d according
di tto th
their
i
size under native conditions; in water, the elecrophoretic
mobility of DNA is independent of molecular weight because
the charge density is constant); however, as the molecules
wander through the pores of the gel, their mobility strongly
depends on molecular weight for sizes of 10 (ten!)-100,000
base pairs
 Electrophoresis of DNA

Topoisomers - Nucleic acids with the same molecular weight

but different shape will also migrate differently under
electrophoretic
l t h ti conditions;
diti for
f example, l DNA can beb linear
li or
circular: the circular DNA is more compact than its linear
topoisomer and therefore travels faster.
faster Different DNA
DNA’ss can
have different topologies (think of a figure 8) and they can be
separated on a gel according to their topological properties
(shape). This is used to study enzymes such as topoisomerase
I and II which play critical roles in DNA replication and are
t
targets
t off anticancer
ti drugs
d
 Electrophoresis of DNA

DNA bending - DNAs of a few hundred base pairs behave

essentially
i as rigid
i i rods; however, certaini sequences (AT-rich
i
sequences), certain chemical modifications induced by drugs
that covalently modify DNA (e(e.g.
g platinated compounds used
to cure various forms of cancer), induce bending in the DNA
itself. These can be separated
p on a ggel under native conditions

RNA structure - RNA molecules are synthesized as single

strands,
d bbut then,
h lik
like proteins,
i ffold
ld iinto complex
l secondary
d
and tertiary structures that are essential for its function.
These structures can be studied and separated by gel
electrophoresis (acrylamide in this case)
 Electrophoresis of DNA

Nucleic acid-protein interactions – gels can be used to study

protein-DNA or protein-RNA interactions. Under native
conditions, the mobilityy of a DNA differs depending
p g on
whether a protein is bound to it or not; if the exchange is slow
compared to the time needed for separation, the two species
will
ill migrate
i t withith different
diff t mobility
bilit andd the
th equilibrium
ilib i
constant can be measured by estimating the amount of DNA
in each band
band. This technique can be used to study all sort of
kinetic and thermodynamic properties of the interaction of
proteins with nucleic acids.