ELECTROBLOTTING ONTO PVDF (IMMOBILON) MEMBRANE

  1. Run protein sample of choice (at least 200 picomoles) on SDS-PAGE.
  2. After electrophoresis, soak the gel in transfer buffer for 10-20 min.
  3. Prepare the PVDF membrane (0.45µm pore size from Millipore) by first soaking it in 100% Methanol for about 3 seconds and then let it rinse in water for 5 minutes to remove the methanol. The membrane must remain wet throughout the procedure. If it does become dry then it must be re-wetted with methanol before it can be used again. The filter is then equilibrated with the transfer buffer for 10-20 minutes (can be done while the gel is soaking).
  4. Assemble the cassette for transfer as follows starting from the negative electrode (BLACK):

    5. sponge wetted in transfer buffer.

    filter paper (Whatman 3 MM) moistened with transfer buffer.

    protein gel (remove bubbles between gel and the Whatman paper.

    PVDF membrane (remove all bubbles).

    moistened filter paper.

    sponge soaked in transfer buffer.

  5. Insert the cassette into the blotting apparatus, making sure that the membrane is facing the positive electrode and the gel is facing the negative electrode.
  6. The protein is transferred over a two hour period at 0.2A or 18V in a Bio-Rad Transblot apparatus.
  7. After transfer, the PVDF is rinsed in water for 5 min to remove and glycine picked up from the transfer buffer.
  8. Place the filter in Commassie blue stain for a 30 min period at room temperature on a tilt table.
  9. Destain the filter for 5-10 minutes and then rinse in water for 5 –10 minutes on a tilt table at room temperature.
  10. Air dry the membrane on a piece of Whatman filter paper. It can be stored by wrapping it in Saran wrap and freezing it at -20oC.

 TRANSFER BUFFER

STAIN

DESTAIN

25mM Tris

5g/l Coomassie Brilliant Blue R

50% methanol

192mM glycine

25% isopropanol

10% acetic acid

20% methanol

10% acetic acid

 

pH to 8.3