2D-PAGE is a technique used to separate complex protein mixtures based on isoelectric point and molecular weight. It involves two sequential steps - isoelectric focusing and SDS-PAGE. In isoelectric focusing, proteins are separated based on their isoelectric point in an immobilized pH gradient. They are then separated by SDS-PAGE based on their molecular weight. The separated proteins can then be visualized through staining and identified through mass spectrometry. While useful for proteomic analysis, 2D-PAGE has limitations such as low reproducibility and dynamic range.

1. Submitted by-
Vivek kumar
M.Sc Microbiology
Bangalore University

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.

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.

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.

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.