We have generated a few phosphomimetics mutants of our protein (constitutively active threonine and serine site). We are wondering about what is the best way to confirm the mutants have active phosphorylation. I believe that there are a few approaches such as immune precipitation, non-raidoactive labeling and phospho tag gels. Moreover, I like to know, if IP is the option, what anti phosphorylation antibody we should use for the detection. Thank you
Please see the attached documents.Article Impact of Phosphorylation and Phosphorylation-null Mutants o...
https://europepmc.org/articles/pmc6916400/bin/nph-225-250-s002.pdf https://academic.oup.com/mend/article/14/10/1583/2751055
The best way to measure phospho-mutants, you will need an antibody that specifically targets the phosphorylated residue. So, you will only detect the fraction of the proteins that are mutated.
In case you don't know the phosphorylated residue you have to go through the mapping of phosphorylated sites to determine whether phosphorylation sites identified are relevant, using mass spectrometry analysis that yields a list of identified sites.
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