You could try a pull-down assay. You immobilize the purified target protein on a resin, flow a cell extract containing the kinase over it, elute the bound proteins, and use mass spectrometry to identify them (control: resin with no protein bound). This can provide a list of candidates.

Agree with Adam Shapiro to find candidate kinase, probably you need to exogenously over express your protein and pull down whatever might be attached. Of course timing must be critical to catch that complex. Also you can use online tools for prediction of phosphorylation sites, if you already know your protein structure even better. For precise determination of amino acids involved in post translational modification (of your target protein) could be examining the phosphorylation state by expressing different fragments of your protein, finally mutation analysis.

Adam B Shapiro Thank you so much for your suggestions.

Tuvshintugs Baljinnyam Thank you so much for your suggestions.

I have some more queries.

1. Is there any alternative to know the interaction in-vivo and find out kinase for this protein?

2. How can I find the location of phosphorylation of this protein in-vivo? whether in ER or in ECM?

If I understood correctly, below will answer both questions:

very broad, unless you have an idea, if you have a candidate and can perform double staining (best is confocal microscope) for both kinase and target protein. Then you can check if they co-localize, but its still really don't tell you they do something to each other, until you do what I suggested previously.

I'm not sure how feasible this is. You might be able to immunoprecipitate the target protein from cells or ECM using an antibody, then use mass spectrometry to identify the site(s) of phosphorylation.

Partially agree with Adam. It would be better if you could over express your gene of interest and perform affinity purification followed by mass-spec. Once you will have got the ms2 file search for PTM (phosphorylation sites). I am sure you have better chance to obtain your phosphorylation sites. Furthermore, to confirm the phospho sites, targeted proteomics can be done. In this process you may also found your potential kinase (candidate) as well. Moreover, don't forget to treat your cells with MG132 which may stabilize your protein; some cases protein goes through rapid degradation upon phosphorylation.

Dear Om,

If you just want to find out if the kinase is intra- or extracellular I have a simple solution for you. Separate the cells from the medium by centrifugation. Lyse the cells and compare ATP depletion rate in lysate and in medium. You can use e.g. the Kinase RR Kit https://biothema.com/shop/kits/kinase-rr-kit/. With this kit you can also characterize the kinase trying different substrates and other conditions.

Best regards,

Arne

Thank you so much Dear, Sayem Miah and Arne Lundin for your suggestions.

May try to make WT and Knockout of your gene of interest in cell and run real-time global kinase activity array. Use PamGene’s global kinase activity arrays. They are more sensitive (1-5 ug proteins only, 10-50k cells), robust, reproducible (