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?

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may soak the membrane in the TBS buffer.

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How about PVDF-membrane? Perhaps this may work better. Have not tried myself.

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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.

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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.

Ginger

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