Dear All,

I would like to use some mTOR inhibitors like OSI-027 to do the cellular thermal shift assay (CETSA) to see the interaction between this molecule and mTOR protein. However, I did not see a very clear thermal shift after treatment with this small molecule. I even tried different concentrations (10 μM, 20 μM, and 40μM) and treatment time (30 min, 1 h, 2 h, 6 h, and 12 h). I think maybe this is due to the high molecular weight of mTOR protein (288.5 kDa). Is that right? Then I plan to transfect 293T cell with pLEX-FHH-MTORKinaseDomain (Plasmid #120708) this plasmid from Addgene and use that cell to do the CETSA so that I can decrease the molecular weight of my target protein. Is that okay?

Thanks a lot!