Hello everyone,

I'm planning a kinase assay and would really appreciate insights, suggestions, or links to protocols/papers that describe similar experiments.

Here’s what I want to do:

• I have a purified His-tagged substrate protein.
• I’ve cloned and am expressing a kinase protein in E. coli (BL21-DE3 or similar). I do not plan to purify the kinase—just use the cell lysate directly after overexpression.
• I want to incubate the E. coli lysate (containing the kinase) with my purified substrate protein and see if it gets phosphorylated.

My specific questions:

My goal is to test whether the overexpressed kinase can phosphorylate my substrate before proceeding to purify the kinase. The substrate protein is stable and already purified.

If you’ve done something similar or know of published methods, I’d be very grateful for your input!

Thanks in advance for your help.

Best, Kaustubh Prakash