2-D electrophoresis is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples.  Two-dimensional electrophoresis was first introduced by O’Farrell and Klose in 1975 2-DGE is a multi-step separation technique in which proteins are solubilized and separated according to charge (pI) in the first dimension using IEF, followed by size (molecular weight, MW) using SDS-PAGE in the second dimension. The separated proteins are stained with coomassie or silver stain to produce a two-dimensional protein reference map.

1. 2d gel electrophoresis

2. INTRODUCTION
2-D electrophoresis is a powerful and widely used method for the analysis of complex protein
mixtures extracted from cells, tissues, or other biological samples.
 Two-dimensional electrophoresis was first introduced by O’Farrell and Klose in
1975
2-DGE is a multi-step separation technique in which proteins are solubilized and
separated according to charge (pI) in the first dimension using IEF, followed by
size (molecular weight, MW) using SDS-PAGE in the second dimension.
The separated proteins are stained with coomassie or silver stain to produce a
two-dimensional protein reference map.

3. FIRST DIMENSION –ISO ELECTRIC FOCUSING
Isoelectric Focusing is an electrophoretic method that separates proteins according to their
isoelectric points (pI).
Proteins are positively charged at pH values below their pI and negatively charged at pH values
above their isoelectric point.
The presence of a pH gradient is critical to the IEF technique.
 In a pH gradient and under the influence of an electric field, a protein will move to the position
in the gradient where its net charge is zero.
Isoelectrofocusing was performed by using Immobilized pH gradient (IPG) strips (7cm, pH 3-10;
Bio-Rad).
IPG strip are made with buffering acrylamide derivatives that contain either a free carboxylicacid
or atertiary amino group that is coplymerised with acrylamide and bisacrylamide.

7. STEP 1 -REHYDRATION PROCESS
Prepare sample in, or dilute in to a suitable rehydration buffer
Pipett indicated volume (125μl ) of each sample as a line along
the edge of a channel in a rehydration tray.
Peel the cover sheet from the IPG strips using forceps.
Then gently place the IPG strips with gel side down on to the
sample.
Overlay each of the IPG strips with 2-3 ml of mineral oil.
REHYRATION
BUFFER -5ML
Urea - 2.5gm
Thiourea -
0.75gm
CHAPS - 0.15gm
DTT - 0.05gm
Bromophenol
blue - 1pinch
Biolyte - 5μl

8. Rehydration tray

9. 2. Pipett indicated volume (125μl ) of each sample as a line along the edge of a channel in a
rehydration tray.

11. IPG STRIP

12. Peel the cover sheet from the IPG strips .

13. Then gently place the IPG strips with gel side down on to the sample

16. overlay each of the ipg strips with 2-3 ml of mineral oil.
Cover the rehydration tray with lid and leave the tray overnight (11-16hr) to rehydrate
IPG strips and to load the protein sample.

17. place a dry clean IEF tray,

18. Pipett 10μl of deionized water to wet wicks

19. Keep paper wicks at each end of the channel to cover electrode
wire

20. using forceps carefully hold the rehydrated IPG strips from the
rehydration tray and blot the tip of the strips on a filter paper to allow the
mineral oil to drain.

21. Transfer the strip to IEF tray, and 2-3 ml mineral oil and close the tray

22. Place the focusing tray in to a protean IEF cell and
close the cover.
Program the protean IEF cell default temp 20◦C
and with a maximum current of 50μA.
When electrophoresis run has been finished,
remove the IPG strips from tray and let the mineral
oil drain.

23. STEP 2-EQULIBRIATION
• Equilibriate the IPG strips in SDS containing buffers.
• It ensures that cysteine are reduced and alkylated, which eliminates vertical streaking.
• Equilibriation bufffer1 contains DTT which reduces sulfhydryl groups.
• Equlibriation buffer II contains iodoacetamide which alkylates the reduced sulfhydryl
groups.

25. Second dimension SDS PAGE
• And then placed on SDS polyacrylamide slab gels for second dimension run.
• The second dimension separation was performed with 12% (w/v) polyacrylamide gel with a
5% (w/v) stacking gel on a mini-Protean 3 electrophoresis cell (Bio-Rad).

26. The gels after electrophoresis, was
incubated in fixing solution in a shaker for
20minutes(10% aceticacid +40% methanol)
wash with milli Q water
staining for visualization of protein spots
Coomassie blue staining solution (0.025%)- 1lit
CBB - 0.25gm
Methanol - 500ml
Acetic acid - 50ml
Adjusted the volume up to 1000ml by adding Milli q
water

28. IMAGE ACQUISITION AND ANALYSIS
After running sds-page gel computer assisted image analysis is done by
IMAGE SCANNER III LAB SCAN 6.0 (GE HEALTHCARE),using
PROGENESIS SAME SPOT software.

29.  Spot detection
 Spot quantitation
 Gel comparison
 Statistical analysis
Each spot on the resulting two- dimensional gel potentially corresponds to a single protein species in
the sample.

30. IDENTIFICATION AND CHARACTERIZATION OF 2D
PROTEIN SPOT
• By amino acid sequence analysis we will get the sequence
data .
• or we can generate peptide mass finger printing by using
mass spectrometry.

31. Thank you