3.5% acrylamide gels and transfer - I need tips (Sep/09/2008 )
Hi. I have to resolve three bands in a SDS-PAGE (95, 190 and 550 kDa). I'm doing 3.5% acrylamide gels followed by transfer to nitrocellulose. The gel is ultra sticky and fragile, I can place it in the transfer apparatus but the problem comes the following day: the gel sticks to the membrane, it's almost impossible to separate from the NC membrane. Have any of you worked with low percentage acrylamide gels? Any suggestions?
may soak the membrane in the TBS buffer.
How about PVDF-membrane? Perhaps this may work better. Have not tried myself.
Yes, I've read PVDF membranes are better in this case. I'll try that. I'll also try the technique described in the paper you sent me.
Was going to suggest the same thing. PVDF is pretty robust, and semi-dry transfer can be done pretty quickly (mine is usually about 15 mins, although your proteins are on the big side, so 30 mins might be better).
I haven't used any gel percentage as low as yours (they must be terribly fragile!) but think PVDF is probably the way to go.