2D GEL ELECTROPHORESIS

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 .

4. step 1 -Rehydration process Prepare sample in, or dilute in to a suitable rehydration buffer Pipett indicated volume (125 μ l ) 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 - 5 μ l

5. Rehydration tray

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

7. IPG STRIP

8. Peel the cover sheet from the IPG strips .

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

10. 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.

11. place a dry clean IEF tray,

12. Pipett 10 μ l of deionized water to wet wicks

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

14. 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.

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

16. 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 50 μ A. When electrophoresis run has been finished, remove the IPG strips from tray and let the mineral oil drain .

17. 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.

18. Second dimension SDS PAGE A nd 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).

19. 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

20. IMAGE ACQUISITION AND ANALYSIS A fter 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.

21. 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.

22. 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.

23. Thank you