STEP 2: First Dimension Gel Electrophoresis

1. IPG strips (pH 3-10) were rehydrated overnight with 350 µl of rehydration buffer (8M Urea, 2% CHAPS, DTT (7 mg per 2.5 ml of rehydration buffer) and 0.5% (v/v) IPG buffer pH 3-10) .

2. IPG rehydrated strip loaded with 100µg proteins in a reswelling tray at room temperature.

3. The focused strips were equilibrated twice (15min each time), Ist equilibration in 50mM Tris-HCl buffer containing 30% (w/v) glycerol, 2% (w/v) SDS, 6M urea, 1% (w/v) DTT and and 2 nd equilibration was performed in a solution containing 4% (w/v) iodoacetamide instead of DTT.

4. Isoelectric focusing (IEF) was conducted at 20ºC with running conditions as follows: Ist hr at 500V , followed by 1000V for 2 nd hr and finally 16 hrs at 3000V.

The IPG strips will be taken out from the apparatus and 2 nd dimensional separation will be perform by SDS-PAGE in a vertical slab of acrylamide.

STEP 3: Second Dimension SDS-PAGE Electrophoresis

The protocol to perform SDSPAGE is discussed in a earlier lecture.

STEP 4: Gel Silver Staining

The protocol to perform silver staining is discussed in a earlier lecture.