I purified kinase and target protein in E.coli and did the invitro phosphorylation assay. Then ran a 6% phos-tag page. But after I blotted the page, the result seemed wired. I cannot sure whether the target protein is phosphorylated or not. if there is a shift, why the band in the control group(un-phosphorylation) is so weak compared with the shifted band. 2 why the kinase inhibitor cannot block phosphorylation. 3. why I add lambda-ppase cannot also ablate phosphorylation.
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Why you run 6% phos tag page , before run you put standardisation by different percentage
Hi. Generally speaking, I think if you use λ phosphatase and still see band, that shouldn't be a phosphorylated band.
Check this paper:
https://www.nature.com/articles/s41388-021-02117-5
Tyrosine phosphate usually (but not always) gives you a shift, and it may be in either direction (yes, that's weird). Ser/Thr phosphorylation usually does not give visible shifts.
What kind of phosphorylation are you after, what type is your kinase?
Do you have the possibility to use phosphorylation site specific antibodies instead, possibly after immunoprecipitating your protein of interest?
Seems the inhibitor is not working as expected and also your phosphatase.
Try dissecting your experimental setup by testing all components. p-Nitrophenylphosphate could be a good control for the phosphatase and try to establish a positive control for the kinase and also for the phostag reagent. Ask the supplier's tech service for assistance, you have paid for it already.
If you are planning to analyze a larger numbers of samples, consider having your own phosphorylation specific antibody made.
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