Last updated: 1/12/12 By: Tim Starr
This protocol describes transferring proteins from an SDS-PAGE gel to a membrane. Three types of membranes are available for transferring: Nitrocellulose, PVDF, and nylon. Generally, nitrocellulose is easiest to use while PVDF has better properties. Membranes can have different pore sizes and some membranes have front and back sides.
PVDF membranes have different sides. Immun-Blot PDVF membrane 0.2µm from BioRad. Keep track of which side is up. PVDF-plus membranes have a shiny side and a dull side because the pores are actually cone-shaped. Small end of cone is shiny side, while large end is dull. On the BioRad roll, the dull shiny side is on outside of roll, dull side is on inside. I try to put the dull side (large end of pores) facing the gel. When I do washes and everything else, I keep this side facing up. According to mfcturer it doesn’t matter as long as you are consistent.
- Make transfer buffer and let it sit for an hour at least to de-gas. Generally better to make the transfer buffer fresh than to use an old stock.
- Try to minimize touching the gel or membrane. Use forceps with the membrane and only touch the edge.
- Cut membrane to be same size as gel. Cut four pieces of 3 mm Whatman filter paper the same size as gel.
- Activate the PVDF membrane. Place in a tray with methanol for 15 seconds, transfer to a tray with water for 2 minutes, and finally place in a tray with transfer buffer for 5 minutes. Nitrocellulose membrane should be soaked in transfer buffer for 5 minutes
- Soak 4 to 8 blotting pads (the sponges) and 3 mm Whatman filter paper (4 pieces of filter paper per gel) in transfer buffer.
- When gel has finished running, crack open the gel cassette using the pry knife. I try to pry off the smaller side of the gel cassette so the gel is resting on the larger cassette.
- Cut off the stacking gel portion of the gel.
- Cut off the bottom part of the gel that is in the groove with the holes
- Pipette about 1 ml of transfer buffer onto the gel to cover the gel.
- Place two Whatman filter papers on top of the gel, making sure there are no bubbles. Pipette transfer buffer onto these filter papers to liberally soak them.
- Flip the whatman filter paper/gel/cassette over onto parafilm or clean desktop. Using the pry knife separate the cassette from the gel/filters. Be gentle. Once you have removed the cassette, pipette another 1 ml of transfer buffer onto the gel.
- Place the membrane (dull side facing gel if using PVDF membrane) onto the gel. Cut a small piece of the upper left hand corner of the membrane (the corner above Lane 1) before placing it on the gel for easy identification later. Make sure there are no bubbles.
- Pipette more transfer buffer onto the gel and place two more Whatman filter papers on top of the gel.
- Put two soaked blotting pads onto the Anode side of the blot module. (This is the box-like side of the module). Place the filter/gel/membrane/filter sandwich on top of the two blotting pads. Remember: Gel is towards the anode (-), membrane is towards the cathode (+), because proteins are coated with negatively charged SDS and will migrate towards the cathode.
- If running two gels at once, put two more blotting pads on top of the first gel/membrane. Then put the second gel/membrane in the blot module box.
- Put enough remaining blotting pads to fill up the anode box. Place the cathode top on the box, squeeze together and insert into the electrophoresis chamber and fasten the clamp.
- Pour enough transfer buffer into chamber so that blotting pads are covered + 0.5 cm above (don’t go all the way to the top).
- Fill the outer chamber with water.
- Connect electrophoresis box to a power supply and run transfer at 30 V for 1-3 hrs or 170 mAmps overnight in the cold room. Note, different voltages and times are required for different gels and membranes. You can check to see whether or not transfer has occurred by opening blot module, gently lifting corner of the membrane to check whether or not the protein ladder proteins have transferred. Transfer buffer recipe depends on type of gel you are transferring from and on the membrane. The following recipe works for PVDF and nitrocellulose membranes and Bis-Tris gels. If using NuPage gels, use the NuPage transfer buffer and follow their instructions.
Transfer Buffer 1 L