Does anyone know an effective buffer mix to use for high current Western transfers? We are successfully using the vendor's premixed buffer to transfer a wide range of protein sizes to PVDF membranes at 1A/25V for 10 min. We get great results with the vendor's expensive buffer.
I haven't been able to find a non-proprietary recipe that works. The best ones we have hold the 25V, but then have a decrease in conductivity (increase in resistance) where they will start at 1A, but drop to 0.4A by the end of the 10 min. The vendor's buffer seems to hold the high current and results in better transfers.
I don't know if it will be helpful to list all the variations I've tried in detail, but they have revolved around modifying a traditional Towbin buffer plus SDS, more glycine/Tris, or MgCl2. These were all various suggestions from people around the department, but I haven't found much published evidence for a buffer under these conditions.
I realize 1A (and no I don't mean 1mA) is a lot of current, but the vendor's system works really well. Any pointers on what I might try/add would be appreciated even if you don't have a worked out protocol.
Edit: Info from the MSDS indicates it has 3 reagents:
Listing of dangerous and non-hazardous components: Proprietary Reagent K 10-20% Proprietary Reagent EB II 5-10% Proprietary Reagent S 1.0-2.5% 7732-18-5 water 50-100% · ..... Solvent content: Organic solvents: 0.0 % Water: 74.8 % Solids content: 25.2 %
[I'm not sure if this MSDS info should be put here, just because I'm looking for someone who has already used a high current buffer they know the formulation for, not a guess to what I have.]