2. Introduction
2D-PAGE is a widely used method for the analysis of
complex protein mixture extracted from cells, tissues,
or other biological samples.
This techniques was first developed by O’Farrell and
Klose in 1975.
2D-PAGE is used in 2 sequential steps-
1. Isoelectric focusing
2. SDS-PAGE
3. Sample preparation
Must select appropriate method to get selected proteins
from cellular compartment of interest.
Must break all non-covalent protein-protein, protein-DNA,
protein-lipid interaction, disrupt S-S bonds.
Must prevent proteolysis, accidental phosphorylation,
oxidation, cleavage.
Must remove substance that might interfere with
separation process such as salts, polar detergents (SDS),
lipids, polysaccharides, nucleic acid.
Must try to keep proteins soluble during both phases of
electrophoresis process.
4. Isoelectric focusing(IEF)
In IEF, proteins are separated by electrophoresis in a pH
gradient based on their isoelectric point (pI).
A pH gradient is generated in the gel and an electric
potential is applied across the gel.
At all pH other than their isoelectric point, protein will be
charged.
The isoelectric point (pI) is the specific pH at which the
net charge of protein is zero.
At its isoelectric point, since the protein molecule carry no
net charge it accumulates or focuses into a sharp band.
5.
6. Immobilized pH gradient and IEF run
Immobilized pH gradients are used for IEF because the
fixed pH gradient remain stable over extended run times at
very high voltages.
This technique has high resolution, great reproducibility
and allow high protein loads.
Isoelectric focusing is run in the same solution that are
used to extract or solubalize the proteins.
The IPG strips with the protein sample must be rehydrated
in the rehydration/ sample buffer during with protein
samples are loaded into the strips.
After the run in IEF cell, the proteins focus as bands on the
strip according to their isoelectric points.
7.
8. SDS-PAGE
SDS-PAGE is an electrophoresis method for separating
polypeptides according to their molecular weight.
This technique is performed in polyacrylamide gels
containing sodium dodecyl sulfate (SDS).
SDS , an anionic detergent which denatures the protein by
breaking disulfide bonds and give negative charge to each
protein in proportion to its mass.
9. SDS linearizes the protein so that they may be separated
strictly by molecular weight.
Protein may be further treated with reducing agent, such
as DTT or TRP to break any reformed disulfide bonds and
then alkalated with iodoacetamide to prevent reformation
of disulfide bonds.
Tracking dye may be added to the protein solution to track
the progress of the protein solution through the gel during
the electrophoretic run.
10. SDS Run
The equilibrated IPG strip is placed on the top of the SDS-
PAGE gel submerged in a suitable buffer and sealed in
place with agarose gel.
An electric current is applied across the gel, causing the
negatively charged proteins move out of the gel and
migrate across the gel.
The proteins separate according to their sizes and therefore
by molecular weight.
It is common to run marker proteins of known molecular
weight in a separate lane in the gel, in order to calibrate the
gel and determine the weight of unknown proteins by
comparing distance traveled relative to protein.
11.
12. Visualization
After electrophoresis the gel is stained to visualize the
separated proteins.
Commonly used stained are Coomassie Brilliant Blue or
SYPRO RUBY or Sliver Stain, different proteins will appear
as distinct spot within gel.
13. Application
To protein can be separated in pure form from spots which
can be quantified and also analyzed by MS.
To study of gene products at molecular level.
To study proteomics.
Provides important information correlating the absence or
presence of individual protein characteristic of specific
clinical conditions.
14. Disadvantages
This technique include a large amount of sample handling,
less reproducibility.
It is also not automated for high throughput analysis. 2D-
PAGE has limited dynamic range.
Difficulty to separate low abundance proteins, acidic and
basic proteins, very large and very small proteins and
hydrophobic proteins.
15. Reference
Rabilloud T, Lelong C. Two-dimensional gel
electrophoresis in proteomics; a tutorial. 2011; page
1829-1841.
J Mammary Gland Biol Neoplasia. The application of
2D gel. 2002. page 385-393.
