Protein + alkali = ?

Michael Sullivan via methods%40net.bio.net (by mlsulliv At wisc.edu)
Fri Feb 2 09:44:45 EST 2007


On Feb 1, 2007, at 3:24 PM, Pow Joshi wrote:

>
> 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).
>

When I was in graduate school, we used to do western blots for  
ubiquitin. For these blots, we would routinely autoclave blots post- 
transfer in transfer buffer. Doing this greatly enhanced the western  
signal, presumably because ubiquitin secondary structure was stable  
enough that it didn't fully denature in SDS-PAGE, or that it easily  
refolded during transfer. In any case, this is another method to  
denature proteins on a blot. I don't have any references off hand,  
but you could probably find something in the ubiquitin literatures  
from the 1980's. I think we kept the blot between 3MM filter paper  
and weighted it down with a metal screen of some sort in a pan of  
transfer buffer (which we poured out of the transfer apparatus. Then  
we just autoclaved on the liquid cycle for 20 min.

Mike


---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)



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