Protein Blotting Techniques

Membrane Preparation

PVDF membrane

1. Immerse the membrane in 100% methanol for a few seconds, until the entire membrane is translucent.
2. Rinse the membrane in deionized water.
3. Transfer the membrane to transfer buffer, and incubate until it is equilibrated (2-3 minutes or until it no longer floats). Do not allow the membrane to dry.

PVDF semi-dry blotting with Bio-Rad Trans-Blot device

1. Pre-chill 100 ml of transfer buffer.
2. Assemble the stack on the bottom (anode) plate as follows:
   • Two sheets of whatman wetted with transfer buffer.
   • PVDF membrane, prepared as described above.
   • The gel.
   • Two more sheets of whatman wetted with transfer buffer.
3. Place the cathode and the top cover onto the stack.
4. Transfer for 15-30 minutes at 10-15V for mini-gels.
5. After transfer, rinse the membrane three times (5 minutes each) with distilled water.

PVDF wet-blotting with Invitrogen device

1. Prepare 500ml of NuPAGE transfer buffer from the 20x stock:
   • 25 ml NuPAGE 20x buffer
   • 50 ml methanol
   • 0.5 ml NuPAGE Antioxidant
   • Deionized water to 500 ml
2. Assemble the transfer stack as follows in the deep (anode) compartment (all components should be soaked in transfer buffer):
   • 3 blotting pads
   • 2 layers of whatman
   • The gel
   • PVDF membrane, prepared as described above.
   • 2 layers of whatman
   • 3 blotting pads
3. Put the top plate on, and slide the assembly into the buffer chamber. Then lock the tension wedge.
4. Fill the blot module with transfer buffer to the top of the blotting pads.
5. Fill the outside of the chamber with room temperature water.
6. Run at 30 Volts for 1 hour.