western blotting IEF gels

Zhengyu Sha zsha at iastate.edu
Mon Jan 11 16:25:36 EST 1993


In <Pine.3.05.9301111440.A25760-a100000 at post.its.mcw.edu> fgarbrec at POST.ITS.MCW.EDU (GARBRECHT) writes:

>I am interested in blotting protein mixtures onto nitrocellulose
>which have been separated by IEF.  The problem is that, as the proteins
>come to their isoelectric point in the IEF gel, they have a net zero
>charge and therefore won't migrate onto the nitrocellulose in the
>electroblotting step.  It occurs to be that there might be a couple
>of solutions: 1) to incubate the IEF gel in SDS containing buffer to
>allow the proteins to get a nice negatively charged coating of SDS
>or 2) to do capillary blotting instead of electroblotting (which I
>don't want to do.

>Has anybody tried this or have any suggestions?  Thanks alot

>Fred Garbrecht
>Medical College of Wisconsin
>fgarbrec at post.its.mcw.edu

Try the 2-D gel protocol and let the protein run just into the SDS gel. Then follow the normal protocol. We have western blotted the 2-D gel without any problems. 



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