Electroblotting on to PVDF (Immobilon) Membrane
Categorized: Protein Purification and Gel Electrophoresis
- Run protein sample of choice (at least 200 picomoles) on SDS-PAGE.
- After electrophoresis, soak the gel in transfer buffer for 10-20 min.
- 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).
- Assemble the cassette for transfer as follows starting from the negative electrode (BLACK):
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.
- 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.
- The protein is transferred over a two hour period at 0.2A or 18V in a Bio-Rad Transblot apparatus.
- After transfer, the PVDF is rinsed in water for 5 min to remove and glycine picked up from the transfer buffer.
- Place the filter in Commassie blue stain for a 30 min period at room temperature on a tilt table.
- Destain the filter for 5-10 minutes and then rinse in water for 5 –10 minutes on a tilt table at room temperature.
- 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