Your problem may be the soaking in methanol. It is the SDS/protein complex
that is transferred (you need the protein negatively charged for transfer,
given the polarity of the electric field). If you remove the SDS by adding
methanol, then you will get very poor transfer. We used to take the gels
straight from the SDS-PAGE plates, and put them onto the nitrocellulose,
then get them into the transfer system as fast as possible, for best
transfer. Adding SDS doesn't help, as it can't complex with the protein in
the presence of MeOH.
Katheryn Resing
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