Protocol : The complete process of 2-D gel electrophoresis is given in the Figure 16.2 and it has followsing steps:

STEP 1: Protein Extraction

1. Tissue or cells were frozen in liquid and ground to fine powder. The powder was mixed with chilled (200C) 10% Trichloacetic acid in acetone with 1% DTT.

2. The tissue suspension was incubated for 1hr at 20⁰C and the mixture is centrifuged at 35000g for 15mins at 4⁰C.

3. Discard the supernatant and carefully dissolve the pellet into the ice cold acetone containing 1% DTT. Incubate the suspension for 1hr at -20⁰C and subsequently centrifuge the the mixture at 35000g for 15mins at 4⁰C.

4. Discard the supernatant and pellet was freeze dried multiple times.

5. 25mg of pellet were suspended in 750µl of lysis buffer (9M Urea, 4% CHAPS, 0.8% ampholyte pH 3-10 and 1% DTT) and incubate for 1hr at 37⁰C.

6. Centrifuge the suspension at 12000g at room temperature and collect the supernatant in fresh tube.

7. Determine the protein concentration by lowry or Bradford method.

Figure 16.2 : Different Steps in 2-Dimensional Gel Electrophoresis. needs redraw from