I am trying to do in vitro phosphorylation for one intersting SR protein. I already have the recombined-protein purified from insects cells and with mass spectra to confirm its phosphrylation sites.
For sure, the purified protein has been phosphrylated, but I am planning to use it as a substrate with kinase to perform in vitro phosphorylation in order to confirm the exact kinase that work on this SR protein.
My current thought is to use a Lambda Protein Phosphatase to remove the phosphorylation of this SR protein first and then incubate it with a specific kinase with ATP and buffer(with phasphatase inhibitor).
The kinases are puryfied from E.coli system and the phosphried SR protein will be checked by pro-q dimand staning and then be used for mass spectra to detect the sites that could be affected.
As I don’t have too much experience about in vitro phosphorylation, do you have any concern about my plan?
1. I am a little worried about the Lambda Protein Phosphatase which might inhibit this in vitro phosphrylation even if with the phasphatase inhibitors
2. Should I express this SR protein again in Ecoli not from inserts which should have less phosphorylation
Thank you for your suggestions!
By the way, I will appreciate any recommended protocol for this assay! Prefer the one without 32P labe.
Unphosphorylated recombinant protein as a starting substrate for phosphorylation by kinases in vitro may work better. One can use cold ATP for in vitro phosphorylation by a kinase and detect the phosphorylation of the protein by antibodies specific to p-Ser or p-Thr through Western blotting.
Dear Mingming your plan sounds fine. I agree with Divaker comments above as far as you get reliable antibodies. Would be even better if you have specific antibodies to different suspected phosphorylatable motives within the peptide. I only used 32P labe to map unknown phosphorylation sites. Just watch out that the removal of those phosphates won't affect the affinity of your peptide for the kinase. Although unlikely, some times previous phosphorylations are needed to "prime" the kinase-substrate reaction.
Best
1. I mean you must use the unphosphorylated protein.
2. After Lambda treatment you can go in two ways - purify SR protein ( i hope it has his tag) or just add phosphatase inhibitor like sodium fluoride
1-2 mM
3. Express the SR protein in E.coli if it possible.
Dear Mingming,
I think that the question comes down to whether you want to just demonstrate that the candidate kinase phosphorylates SR protein in vitro or the candidate kinase is "THE" kinase that phosphorylates SR protein in a relevant biological context.
If the prior, then I think your strategy is fine. I agree with Andrei that after treating SR (from insect cells) with phosphatase and purified it, you want to include phosphatase inhibitor (e.g. NaF) in your kinase assay buffer. If you can manage to express SR protein in E.coli, co-expression with an alkaline phosphatase is an option to render you unphosphorylated SR through purification directly.
If you want to pin down the candidate kinase to be "THE" kinase that phosphorylates SR, you probably need to follow up with some genetics study (e.g. knock-out/knock-down).
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