Hi All,
I have a protein of interest, X, and it has a single phosphorylation site identified. Another protein A might dephosphorylate X. I want to knock down A, and quantify the change of phosphorylation on X. The problem is I don't have antibody against the phosphorylated X. Is there any other way that I can detect the phosphorylation level on the protein? Thank you!
You could try a biochemical approach, such as measuring the rate of kinase activity, ATP --> ADP + Pi conversion. But that would be far more technical, difficult, and less direct of a measurement then using a phospho-antibody. You could try to get a trial / sample aliquot from a vendor, that is your best way forward.
Good luck!
You could try immunoprecipitation with an antibody specific for your protein X and afterwards do a western blot with an anti-phospho antibody. This way you do not need an antibody against the phosphorylated protein X.
Best regards,
Christian
Try https://labchem-wako.fujifilm.com/us/product/detail/W01N40AAL-107.html
Going to be challenging...how much material do you have? You could try pSer/pThr or pY combined with a protein specific IP...probably your best option.
Quite simple if your protein genuinely has a single phosphorylation, much harder to link to your phosphatase if there are more phospho sites (quite possible); if there are more, the contribution of your site to the whole protein phosphorylation might be lost in the noise. Ditto for the Phos-tag approach suggested above.
If you have enough material, mass spec might be your best option, although whether it will be quantitative enough to test phosphatase X contribution has to be empirically tested.
Good luck!
Hello,
You can use the radioactivity labelled ATP (P32 or P33) in a in vitro kinase assay. it required the recombinant protein of your kinase and protein of interest.
see https://www.ncbi.nlm.nih.gov/pubmed/?term=mamadou+amadou+diallo for more details.
Do the Mass spec/MALDI, Rapid and accurate, if you have pure phosphorylated protein and its reference
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line