I have a protein SRPK1 ( Serine-arginine protein kinases) and I want to check If the SRPK1 protein phosphorylate to SRSFs ( Serine and arginine rich splicing factors ) by don't use antibody and western blot
what Is there a method that easy and cheap?
I want to Kit or assay for prove it. Please Pointing the light to me.
Thank you sir.
by NK (Nopkamol Kanchanangkul ___Biochemistry and Molecular biology)
Hello, see info below may be useful for you.
Good luck!
One straightforward way to answer the question, if you have access to it, is intact protein mass spectrometry. You would need both SRPK1 and each SRSF protein in purified form. Mix SRPK1 and MgATP with SRSF. If SRSF is phosphorylated by SRPK1, the mass of SRSF will increase by 80 Da for each added phosphate. This analysis can be done by a commercial lab, or by a specialized academic mass spec support lab.
To further determine the site(s) of phosphorylation, you could proteolytically digest the SRSF and use mass spec to look for the phosphorylated peptides.
When purifying the SRSF, you should try to use E. coli expression, if possible, so that the resulting protein will not already be phosphorylated.
https://www.rndsystems.com/products/universal-kinase-activity-kit
https://www.rndsystems.com/resources/articles/methods-detecting-protein-phosphorylation
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