Is 20% methanol sufficient to fix protein in gel? - (Apr/20/2007 )

by accident, one of our PhD students failed to start tank blotting, so the 2D gels were not blotted overnight but in contact with transfer buffer containing 20% methanol; diffusion of polypeptides would have occured or is the 20 % (v/v) methanol sufficient to fix polypeptides, and diminishs/prevents diffusion?

(meanwhile we have blotted the gels and got spots on the blots, however, at the moment I cannot say if they are depositioned by diffusion drift); any suggestion is welcome!

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the methanol is not a fixative. it is supposed to aid in the transfer by stripping sds off of the protein being transferred. it also helps reduce swelling of the gel during transfer.

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thanks mdfenko but from your answer there arises another question: in our Wb transfer buffer we add beside methanol SDS; and should there be no SDS to cover polypeptides with negative charge to guarantee transfer of proteins to the anode (as in SDS PAGE)? stripping SDS by methanol would be contraproductive...

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seems like a contradiction, doesn't it?

but...

a little sds in the transfer buffer helps to get larger proteins out of the gel, the methanol will still strip the sds from the protein (it's not necessarily completely removed).

removal of the sds will allow the protein to bind to negatively charged matrices, like nitrocellulose. it will also allow for hydrophobic binding to pvdf.

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when you mean little SDS, which percentage do you mean ?

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sds in the buffer should not exceed 0.05%. this will help in the transfer of less soluble proteins, according to the "protein blotting handbook, fifth edition" by millipore (pp 14-15).

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