Hello,
I am working with a kinase domain of a membrane tyrosine kinase receptor. This protein undergoes auto-phosphorylation upon binding of a ligand. So is there any assay available by which I can perform in vitro phosphorylation of the purified kinase domain?
Note that the expression system I am using is E.coli.
Incubate the protein with Mg2+ and radioactive ATP in which the gamma phosphate is 32P or 33P. Separate the protein from the excess ATP (such as by SDS-PAGE or with a spin column). Measure the radioactivity in the protein by autoradiography or liquid scintillation counting.
or
Incubate the protein with MgATP. Perform a Western blot with anti-phosphotyrosine antibody.
or
If you have a lot of the protein, you could measure production of ADP by the autophosphorylation reaction, using a sensitive method, such as the ADP-Glo assay kit.
Adam nailed it, one thing I'd like to note about the radioactive assay is that phosphorylation is a labile post-translational modification and 32p through autoradiography or scintillation requires some optimisation. Always keep your sample cold and run any gels on ice.
You can also use affinity chromatography using Gallium IMAC to purify phosphorylated proteins. There are multiple protocols about that online.
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