Western blot of basic proteins

Robert Slany rslany at leland.stanford.edu
Tue Oct 17 02:11:34 EST 1995


Hi netters,
I've to blot a very basic (pHi>10!!) and large 250kDa protein. 
Without SDS in the blotting buffer the protein will be positively 
charged and therefore transfer in the wrong direction. 
(Or presumably not at all, since some SDS will initally stick to 
the protein and make it go into the direction of the anode, then 
the methanol will strip of the SDS and the transfer direction 
will turn around)
Whatsoever, I get a transfer if I do the blot semidry in the 
presence of 0.05% SDS. My problem is that the protein doesn't 
stick very well to the membrane in the presence of SDS, so I 
loose a lot of my detection sensitivity. I've heard of a method 
of transferring in 10mM CAPS buffer at pH 11. Does anyone have a 
protocol for this??
A thousand thanks for your suggestions.

Robert

rslany at leland.stanford.edu




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