Anti-serine phosphatase

hadi al-hasani hadia at bdg10.niddk.nih.gov
Thu Mar 6 23:06:46 EST 1997


The most reliable method to check the phosphoamino acid  composition
is still: 
1. phosphorylate your sample w/ 32-P (metabolic label/in vitro
phosphorylation)
2. run SDS-PAGE , isolate band of interest  and elute tryptic peptides
3. hydrolyze sample in w/ 6N HCl, 110 dec.C, 1h (if you use 1 N KOH,
55 deg. C, 1 h, pSer and pThr should be gone...) 
4. analyze w/  2 or 3 dimensional thin layer
chromatography/electrophoresis

Well, it's a hell of a work but it works!  
Concerning the nice control protein: Why not make your own control
using a commercially availiable PSK, lets say PKC and a substrate in
vitro..? But then you still need a negative contol...( there are also
some PTKs on the market...) 

On 6 Mar 1997 12:27:09 -0500, iayork at panix.com (Ian A. York) wrote:

>
>I need to see if a protein is serine-phosphorylated.  I'm going to use
>Sigma's anti-phosphoserine antibody (IP my protein, then blot it and probe
>with the anti-ps), and I'm aware that it's pretty pathetic.  Does anyone
>have a suggestion for a nice control protein?  Ideally it would be
>moderately abundant, constitutively serine-phosphorylated, easily
>immunoprecipitated with a readily available antibody, and not either 25 or
>50 kDa, because if it is the antibody on the blot will overlay it. 
>
>Also, if anyone has any tips for making the anti-phosphoserine antibody
>more reliable, I'd appreciate hearing about it.
>
>Thanks.
>
>Ian
>
>-- 
>      Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
>      "-but as he was a York, I am rather inclined to suppose him a
>       very respectable Man." -Jane Austen, The History of England




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