Blotting onto PVDF
Shaun D. Black
shaun at uthct.edu
Mon Aug 3 11:12:04 EST 1998
pdxkgs at pdn1.gene.nottingham.ac.uk (Karen Spink) wrote...
>Has anyone had any problems Western blotting onto PVDF in CAPS buffer? I am
>trying to get a sequence on my protein and it appears that the efficiency
>of transfer is letting me down. Could it be the buffer, (10mM CAPS, 10%
>methanol, pH 11) ? I have had no problems blotting onto nitrocellulose
>using tris-glycine buffers.
Why not just blot to PVDF with Towbin buffer (tris/Gly)? We do this quite
often to determine a sequence, and it works just fine. I'm always a bit
concerned about the pH 11 of the CAPS system, as it might promote peptide
chain scission, deamidation of Asn/Gln, etc. You'd think that all the
Gly would be a problem with the Towbin system, but if you wash the blot well
after staining (which you'd likely do anyway), essentially all the Gly is
gone by then. We never see background Gly peaks on cycle 1 of sequencing.
I hope this helps you a bit.
= Shaun D. Black, PhD | Internet e-mail address: shaun at uthct.edu =
= Dept. of Biochemistry | University of Texas Health Center at Tyler =
= Tyler, TX 75710 | World Wide Web: http://psyche.uthct.edu =
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