Protein + alkali = ?

Pow Joshi via methods%40net.bio.net (by pow.joshi At gmail.com)
Thu Feb 1 16:24:56 EST 2007




On 1 Feb 2007 01:45:52 -0800, WS <novalidaddress At nurfuerspam.de> wrote:
> 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?

I do believe the likelihood of serine/threonine phosphorylation, even if such is predicted as "unlikely"; ....considering that the protein itself is a kinase, would there be an autophosphorylation site?....since the alkali would actually hydrolyse the phosphate group .... that would explain why the peptide antibody does'nt work before the hydrolysis (since there a phosphate group present) and the whole protein antibody does...
I would'nt know much about o-glycosylation though .....I wonder if there's a way for you to check either of the possibilities with anti-phospho antibodies/ or by glycosidases .... and yes, if it is just unmasking of the epitope, as DK says, perhaps you could use other denaturing agents? (I am not too sure if acid denaturation removes any putative phosphate group).

best
pow

 
> Wo
> 
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