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Re: detection



At 16:31 15/03/99 +0100, you wrote:
>I found that some companies sell kinase detection kits based on 
>antibody detection. The reaction are performed in microtiter plates 
>and the colored products are quantified by ELISA readers. 
>Do you that it has sense to raise antiphosphoserine antobody for 
>kinase inhibitors testing?
>
>
>
>Vladimir Krystof
>---------------------------------------------------
>Laboratory of Growth Regulators
>Palacky University
>Slechtitelu 11
>CZ-78371 Olomouc
>Czech Republic
>
>phone: ++420 68 5222487
>fax:   ++420 68 5221357
>
>http://prfholnt.upol.cz:81/growthreg
>

I think that it is true to say that the paucity of publications using
antiPS/PT antibodies bears mute testimony to the fact that they do not work
very well (if at all)!!!! There is a  simple explanation for this compared
to antiPY.  The phenolic ring+ PO4 of P- Tyr gives it good antigenicity
compared to PS/PT since it is longer and sits well in the antibody binding
pocket-hence the usefulness of antipY antibodies.  This is not the case
with PT/PS where there is just a carbon chain and a PO4. That's why PS/PT
peptides bind poorly (or not at all) to SH2 domains compared with the
cognate PY peptide. 

Our own experience: we made antiphosphoserine antibodies in rabbits about 8
years ago.  On Elisa testing they bound immobilised phosphoserine on plates
but on specificity testing using analogues we found they were hopeless
since they were easily displaced by phosphate and ATP.  Naturally, they
bound antiphosphothreonine as well!!

The only antiPS/PT antibodies that work with any degree of satisfaction are
ones made against phosphopeptides -vide the number of publications using
antiphosphoMapK and similar and the commercial success of these!!!
Similarly,  we used a antiPhosthothreonine antipeptide antibody against our
favourite protein (raised against a phosphopeptide) and successfully
identified a phosphorylation site.

So, if you want to make a  PS/PT antibody use a phosphopeptide.