how to find out inhibitor target

[yi198720022004]

Hi,

That's say if I found an inhibitor to a pathway. Among many points in the pathway, how can I find out that which point the inhibitor targets. Can I perform a gel shift assay?

[magicsiew]

Can use microarray to see the gene expression pattern, to identify which gene is inhibited.

[JackBean]

question is, whether he talks about enzyme or signaling pathway

[canalon]

Depends of the nature of the inhibitor, and the strength of the link between the inhibitor and the target.
For protein/protein interaction, the SPINE technique appears good on paper (it is a bit more tricky in my experience, but I set myself for difficult conditions in the first place). It consist in labeling your inhibitor protein (his tag, strep tag) and regulate expression then fix your cells with PFA and run on a column designed to attract your labeled protein. Then elute and boil and you have your target and your prey in the mix.