be in either a native ora denaturing mode

IEF is carried out in an acrylamide gel and can run

Native IEF retains protein structure and enzymatic activity. The use of native IEF, however,

not soluble at low ionic strength or havve

is often limited by the fact that many proteins are

IEF is performed in the presence of

low solubility close to their isoelectric point. Denaturing
dissociates proteins into individual subunits and abolishes
high concentrations of urea, which
urea is the denaturant of choice, as this
secondary and tertiary structures. In denaturing IE,
not otherwise soluble under IEF conditions.
uncharged compound can solubilize many proteins
a stable, continuous pH gradient
between the anode and
Two methods are used to generate
and polycationic mol-
carrier ampholytes, a mixture of polyanionic
cathode. One method uses
is applied
with closely spaced pI values. When a voltage
ecules that carry multiple charges
with the lowest pI (and the most
across a carrier ampholyte
mixture, the carrier ampholytes
carrier ampholytes with the highest pl
toward the anode, and the
negatively charged) move
move toward the
cathode. The other carrier ampholytes align
(and the most positively charged) the pH of their environment.
their pl and will determine
themselves in between according to
method uses immobilized
to 10. Another
The result is a pH gradient from pH 3
continuous
groups to a polyacrylamide gel
formed by covalently grafting buffering
pH gradients strips
-

a stable pH gradient
that can be
groups generates
backbone. A gradient of different buffering
tailored for different pH ranges and gradients.

2.2.4 Two-dimensional gel electrophoresis

of samples by 'one
form of protein gel electrophoresis is the analysis
The most common

are added to sample wells

at the top of the
dimensional gel electrophoresis'. Protein samples
is applied, the proteins move down through the gel matrix,
gel. When the electrical current
are easily compared to each other
lanes' of protein 'bands'. Bands
creating what is called thickness of protein
The intensity of staining and
after staining or other detection strategies.
abundance. The positions of bands within
their respec
bands are indicative of their relative
sizes. Though this is a common electrophoretic method, can
tive lanes indicate their relative
the physicochemical properties
separation of protein molecules having
same
not be used for
cannot be also used for separation and
fractionation of
such as pI and molecular weight. It
For separation and analysis of hundreds to
complex protein mixtures from biological samples.
thousands of proteins in powerful electrophoretic method called two-dimensional gel
one gel, a
a mixture
electrophoresis is used. Two-dimensional gel electrophoresis is based on separating
of proteins according to two properties, one in each dimension. The first dimension separates

to their native isoelectric point (pI) using a form of electrophoresis called

proteins according
isoelectric focusing (IEF) and the second dimension separates by mass using SDS-PAGE. TWo
dimensional gel electrophoresis provides the highest resolution for protein analysis and is an

in proteomic research, where the resolution of thousands of proteins on

important technique
a single gel is sometimes necessary.

First-dimension separation: This is performed by denaturing IEF. Using this technique, proteins
are separated on the basis of their pl. In IEF, proteins are electrophoresed into a pH gradient.
As the proteins move through the gradient, they encounter a point where the pH is equal to
heir pl and they stop migrating. Because of differences in pl, different proteins stop (focus)
at different points in the gradient.

Equilibration: A conditioning step is applied to proteins separated by 1EF prior to the second
dimension separation. This process reduces disulfide bonds and alkylates the resultant sulfhydryl
 are coated with SDS for separation
residues. Concurrently, proteins
groups of
the cysteine

on the basis of mass.

SDS-PAGE. Proteins that have been

is performed by
separatlon: This part
Second-dimenslon
based on their size or
in a second dimension
can then be separated
on an IEF gel
separated a second polyacrylamide gel
is placed lengthwise on
accomplish this, the IEF gel
mass. To
the proteins migrate from the
IEF gel
SDS. When an electric field is imposed,
saturated with resolution of pro-
mass. The sequential
and then separate according to their
Into the SDS gel cellular proteins.
excellent separation of
their charge and mass can achieve
teins by
SDS-PAGE
Isoelectric focusing

High masSS

10
Loading of gel strip|
pH gradient on second gel

Separation in first
dimension (by charge)

Low mass

Separation in second
dimension (by size or mass)

involves
dimension in a 2D gel electrophoresis
Two-dimensional gel electrophoresis. The first
Figure 2.8 isoelectric focusing (IEF). IEF works by
to their isoelectric point (pI) by
the separation of proteins according laid horizontally
isoelectric focusing gel is then
within a pH gradient. The
applying an electric fleld to protein electrophoresis.
gel, and the protelns are separated by SDS polyacrylamide gel
on a second, slab-shaped Horizontal separation
direction of the isoelectric focusing.
SDS-PAGE is performed perpendicular to the appear on
bands that
reflects differences in mass. Thus, the
reflects differences in pl; vertical separation
their lisoelectric points and then by size.
the gel have been separated first by

2.2.5 Sample visualization in the gel

After the electrophoresis run is complete, the gel must be analyzed qualitatively or quantita-
to determine
tively. proteins are not directly visible, the gel must be processed
Because most
the location and amount of the separated molecules. Once protein bands have been separated
by gel electrophoresis, they can be blotted (transferred) to a membrane for analysis by west
ern blotting. Alternatively, they can be visualized directly in the gel using various staining or

detection methods. Proteins are often visualized by staining (gel staining).

Coomassie brilliant blue (the lower detectable limit about 0.1-0.5 ug of protein) is the most

widely used dye which binds to proteins but not to the gel itself. The dye binds nonspecif
cally to virtually all proteins. In acidlc bufer conditions, Coomassie dye binds to basic and
hydrophobic residues of proteins, changing in color from dull reddish-brown to intense blue.
Slver staining is the most sensitive method for permanent visible staining of proteins in po-
lyacrylamide gels. In the case of silver staining, silver ions are reduced to metallic silver on
the protein, where the silver Is deposited to give a brown-black color. The silver ions intere ct
a

and bind with carboxylic acid groups (Asp and Glu), imidazole (His), sulfhydryls (Cys)
amines (Lys). The silver staining is about 100 times more sensitive than Coomassie brt
blue, with a lower detection limit of about 0.5 ng of proteins.

Electrophoresis
 Gel Gel staining with silver stain
staining with Coomassie dye

Estimating the molecular weight of a protein

SDS-PAGE is a reliable method for estimating the molecular weight of an unknown protein.
The molecular weight of a protein can be estimated by comparing the distance it migrates
thirough a gel with the distances that proteins of known molecular weight migrate. Because
shape afects the mobility of a molecule through a gel, all the molecules in one gel must have
similar shapes for valid comparisons.
In SDS-PAGE, SDS denatures proteins by forminga stable
complex that removes most native
Folded structure. The electrophoretic mobility of a protein coated with SDS on an SDS-PAGE
is inversely proportional to the
logarithm of its molecular weight. When a set of standard
teins of known molecular
pro
weight (called molecular weight markers) are run alongside samples
in the same
gel, they separate into a series of bands and provide
reference by which the
a
molecular weight of sample proteins be
determined. The molecular weight markers are
can
used togenerate a curve correlating molecular weight and migration in the gel, from which
the molecular weight of the unknown
sample proteins can be determined.
To determine the molecular weight of an unknown
sample protein, we should separate the
sample on the same gel with a set
of molecular weight markers.
After separation, we should
then determine the relative
migration distance (R,) of the molecular
unknown sample protein. is defined as the
weight markers and the
R, migration distance of a protein band divided
by the migration distance of the ion front. Because
the ion front can be
mobilities are normalized to the tracking dye that difficult to locate,
migrates only slightly behind the ion
front
R
Migration distance of protein band
Migration distance of dye front

Based on the values obtained for the molecular

weight markers, the log of the
weight of an SDS-denatured polypeptide and its relative molecular
migration distance (R) is
a graph. If proteins are fully denatured and the plotted into
gel percentage is
appropriate for the molecular
weight range of the sample, the plot will be linear. The standard
curve is
molecular weight values because, at high molecular weight, the sigmoid at extreme
sieving effect of the matrix is
so large that molecules are unable to
penetrate the gel. At low molecular
effect is negligible and proteins migrate almost freely. To weight, the sieving
determine the molecular
the unknown protein band, we interpolate the value from this
weight of
graph.
The molecular weight
or relative molecular MW (kDa)

mass (Mr) is defined a s 250

molecule's
the ratio of a
mass to that of 1/12
150
the mass of carbon-12
( C ) . Since molecular
-0.9
100 vhere, Y
weight is a ratio, it is
dimensionless-it has 75
ne associated units.
50 Unknown
Molecular mass, not a
protein
s defined as the
25
15
mass molecule

anc 9enerally
expressed in daltons

(Da). Dne can say that

myosin has a molecular
Molecular
weight
Unknown
sample
R
mass of 205000 Da or markers protein
molecular weight of
Figure 2.9 Estimating the molecular weight
205000.
of an unknown protein by electrophoresis. The
electroono
reticmobility of many proteins in SDS-PAGE is inversely proportional to the
logarithm of their molecular
weight (MW). First, molecular weight markers and unknown sample protein are run on a SDS-PAGE. The
migration distance is measured from the top of the resolving gel to each molecular
and to the dye front. For each band
weight marker band
of the molecular weight marker, the R, value is calculated. This step
is repeated for the
unknown protein band in the sample. The
log molecular weight value is plotted as a
function of R. By using the formula for the
slope of the line, y
=
mx + c, themolecular weight of the
unknown protein is determined.