Protein + alkali = ?

Michael Sullivan via (by mlsulliv At
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.


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

More information about the Methods mailing list