In which combination concerning buffer and gels do you use your system?

I know the system from BioRad and it is working when you take their gels and buffers and in best case also their pre-wet stacks. I think this system is strongly optimized from their side with their compounds that you have to stick 100% on this and don't use anything homemade at all.

I am using a Bio rad semi dry system. You have to experiment a bit on the tranfer time and stain both the membrane and the gel in the end of electrotransportation to determine the optimum transfer time (with the less protein left-over in the gel)

Good luck

http://www.yashimachem.co.jp/iPadweb/%E3%82%A2%E3%83%88%E3%83%BC/WesternBlott-kotsu2006.pdf

 introduces and recommends the discontinuous buffer system with semi-dry blotting.

-- Summary from here--

the sandwich image is :

(+) fp(A)x2-fp(B)x1-PVDF-Gel-fp(C)x3 (-)

here, (+),(-) :electrodes; fp(A),fp(B),fp(C) : filter paper immersed in buffer A, B or C;

PVDF: PVDF membrane immersed in buffer B; Gel: PAGE-gel immersed in buffer B; buffer A:300mM Tris, 5% methanol; buffer B:25mM Tris 5% methanol; buffer C:25mM Tris, 40mM 6-aminocaproic acid, 5% methanol.

condition: constant current 2mA/cm2, 30-40min.

when blotting efficiency is low, add 0.01-0.02% SDS in buffer C

-- to here --

Good luck

Thanks, we have Thermo Scientific's Fast Blot G2 on a 2 month trial. We have also used BioRad's semi-dry system and we don't get as strong a signal as we would with a wet transfer using both and yes there is still protein left on the gel and not as much as transferred to the membrane. Yes we have been using our own Tris-glycine buffer with 10% ethanol. I guess I will wait for Thermo to send their buffer and try it once again.

I have always seen better results with Wet Transfer. Having said that I also use the BioRad Semi Dry. We make all our own ingredients and unsure whether that effects it or not, I find you need to optimise for the transfer based on MW as different sized proteins transfer at different times.

Thanks Russell, I agree, I think that if you want to be sure that all protein is transferred, a wet transfer is necessary, usually there is no bands remaining on the gel and the membrane stains stongly with Ponceau, the same cannot be said for a semi-dry transfer using either the Thermo Fast Blot G2 or the Bio Rad System.

I don't think you can really say like this, Jason as I tried in the lab with BioRad system sticking 100% on it and even with a 7 min transfer it was working with samples that no protein was remaining in the gel. But this was only working when using 100% BioRad compounds (gel, wet stack membranes), with my own membrane and own buffer there was a minimal amount of protein remaining in the gel after 30 min of transfer time with a lower voltage (15 V, 30 min) compared to 24 V, 7 min.

Thanks Daniella; Thermo have now sent me their buffer to try. I will report back.