I do not have purified or recombinant kinase or the substrate proteins of my interest. So I transfect cells ((HEK293)/MCF7) with tagged constructs FLAG-tagged for substrate of interest and HA-tagged for the kinase of interest and then perform Immunoprecipitation with the respective antibodies against the tags(anti-FLAG and anti-HA) to pull down the proteins and use the beads as the kinase source and substrate protein. Can anyone suggest a protocol compatible with this.
I'm not sure how you are doing the assay (labeled ATP with autoradiography?). However, you could do it this way. Make lysates in kinase buffer, add hot ATP. Remember that Km values range from 10 - 150 uM for many kinases, so you must supplement the hot ATP with cold to make the final concentration of ATP in the assays in this range. Do the kinase reactions two way: Pre-absorb with either neg control or HA-tag beads; do the reaction; then immunoprecipitate the FLAG antigen and analyzed for phosphorylation. If the antigen is targeted by your HA-kinase, you should see a greater signal in the control absorbed lysates. You could also use specific inhibitors (if available) as controls, or even do the phosphorylation reaction in the live cells. However, the latter uses a lot of radioactivity.
May be we can help you witg iut kinome activity array from Pamgene which is very inexpensive with current proteomic techniques.
https://www.pamgene.com/en/Services-Kinase.htm
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