Biotechnology and Bioinformatics (1+1)

Transferring proteins from the gel to nitrocellulose membrane

1. What man no. 1 filter papers of about six numbers are cut and used as absorbent paper and the nitrocellulose membrane is cut to the exact size of the gel.

2. The membrane and filter papers are soaked in the transfer buffer before stacking for transfer.

3. On a solid plastic platform, two pieces of wet absorbent paper are stacked.

4. Another absorbent paper is placed on the gel in the plate and slowly the gel is transferred on to the stack, placing the gel side up.

5. The wet nitrocellulose membrane is placed on the gel, aligning the edges and the remaining absorbent paper is kept on the stack. (Gel and membrane are sandwiched between stacks of filter paper.)

6. All the air bubbles between the layers are removed by rolling a pipette from the center to the edges.

7. The transfer sandwich is positioned to the electro blotter in such a way that membrane is near the positive electrode. The plates of the apparatus are screwed.

8. The transfer buffer is poured into the transfer apparatus and the electrodes are connected and the power supply is set to ~ 60mA. Transfer is done for one hour.

Note: More transfer tips:

i) The gel needs to equilibrate for 15-30 minutes in transfer buffer . Failure to do so will cause shrinking while transferring, and a distorted pattern of transfer .

ii) Avoid touching the membrane with your fingers; use tweezers instead. Oils and proteins on the fingers will block efficient transfer and create dirty blots.

iii) After sandwiching the gel and membrane between paper air bubbles between the gel and membrane can be removed by rolling them out with a pipette or 15ml tube, or by assembling the sandwitch in a dish of transfer .

iv) Add buffer to prevent formation of bubbles in the first place . Wear gloves !

v) Make sure the filter paper and membrane are cut to the same size as the gel .

Large overhangs may prevent a current from passing through the membrane in semi-dry transfers .