5. Place another blotting sheet (already saturated with transfer buffer) on the top and remove trapped air bubble by rolling test tube or glass rod.

6. Finally keep cathode plate (usually red colored) and tight the transfer cassette by four screws. The whole transfer assembly is given in the Figure 15.2.

Figure 15.2: Steps in electroblotting of protein on the membrane.

C. Transfer protein from gel to the membrane: Place the transfer cassette in the tank filled with the transfer buffer. Connect the transfer apparatus to the power supply unit and apply constant voltage (100 volts for 1hrs). After transfer, disassembly the whole cassette and carefully remove transfer membrane and check the protein transfer by ponseau S stain. Use a pencil and label different lanes.

STEP 4: Blocking: Wash the membrane with distilled water to remove any remaining ponseau S stain. Put the membrane in blocking buffer containing 5% skimmed milk (for detection of cytosolic protein) or 5% Fat free BSA (for detection of phosphor protein) for 30 mins.

STEP 5: Probing: In western blotting, probing can be done in two different ways.