Protein + alkali = ?

WS via methods%40net.bio.net (by novalidaddress At nurfuerspam.de)
Thu Feb 1 04:45:52 EST 2007


Dear all,

we try to detect a certain protein with a peptide antibody on
westerns.

At first, we do not get any signal, but after treating the membrane
(PVDF) with an alkaline (pH12-13) solution for a short time, the
protein may be detected in the reblot and there is a very strong
signal with 100ug of total protein per lane (4mm) . With another
antibody raised against the complete protein, there always is a
signal. Both ABs are rabbit ployclonals.

I have checked the peptide sequence with NetOGlyc and NetPhos: There
are two potential O glycosylation sites, a serin and a threonin, which
are predicted to be unlikely to be phosphorylated. The protein is
known to be a cytoplasmic protein kinase.

What's happening there?

Wo



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