1. When the SDS-PAGE is approaching the end of its run, rinse the plates with laboratory grade distilled water followed by wipe off any beads of liquid that adhere to them with nonabsorbent tissues using methanol.

2. Wearing gloves cut around 4 pieces of whatmann 1 mm paper and one piece of PVDF membrane to the exact size of the SDS-PAGE gel (if the whatmann paper or filter is of size larger than the polyacrylamide gel there is a good chance that the overhanging edges of the paper and the filter will touch causing a short circuit that will prevent the transfer of protein from the polyacrylamide gel to the filter). Mark one end of the filter with a soft lead pencil or cut the filter slightly on the lower left side.

3. To activate the PVDF membrane, soak it in absolute methanol for 1 minute. Thereafter, soak the PVDF membrane in transfer buffer.

4. Soak the pieces of whatmann paper in a shallow tray containing a small amount of transfer buffer.

5. Wearing gloves, set up the transfer apparatus as follows:

I) Place around 2 pieces of soaked whatmann paper on the bottom electrode.

II) Now very carefully place the soaked PVDF membrane on top of the whatmann paper carefully.

III) On top of the PVDF memberane carefully place the gel preventing entrapment of any air bubble.

IV) Place 2 pieces of soaked whatmann papers on top of the gel (At each step, stack the sheets on top of the other so that they are exactly aligned.) Using a spacer or a glass pipette as a roller, squeeze out air bubbles if any.

V) Place the upper electrode on top of the stack. Connect the electrical leads. Apply a constant current of 2.5 A for 7 min.

VI) After 7 min turn off the power supply and disconnect the leads. Disassemble the transfer apparatus from the top to downward, peeling off each layer in turn. Place the memberane in a tray containing Ponceau S for staining.

VII) Wash the membrane with distilled water. Block the non-specific binding by incubating the membrane with 1xTBS containing 2% BSA w/v for 1 h at room temperature.

VIII) After blocking, wash once with 1xTBS containing Tween 20 and incubate with primary antibody overnight at 4ᵒC. The antibody should be diluted with 1xTBS with 0.5% BSA (w/v) according to the supplier’s instructions. Once the incubation time is over, remove the primary antibody and wash the membrane three times with 1 x TBST

IX) After washing, incubate the membrane with secondary antibody which is conjugated with HRP (Horse Radish Peroxidase) and diluted in 01xTBS with 0.5% BSA for 1 hr at room temperature. Following the incubation, remove the antibody solution and wash three more times with 1 x TBST

X) Visualize protein expressions using the chemiluminescence detection substrate (Millipore) using chemidocumentation system.