I think with INSR you have to consider two things: Basically the measuring of the kinase activity is quite straight forward as soon as you have isolated the kinase from the tissue. There are quite a number of well established assays available for measuring in-vitro tyrosine kinase activity, the "classic" approach would be based on radiolabelled ATP. If you have access to an isotope lab this would be one possibility for doing a real robust and well established activity assay.

The other point is the isolation of the endogenous protein - as INSR is an integral membrane protein, its reproducible isolation should be well established. You may consider to use some commercially available kits for membrane protein isolation (e.g. from Pierce) which allow the isolation of membrane proteins in their native form. INSR may then be immune-precipitated form the membrane protein fraction and the IP subsequently used for in-vitro activity determination.

Alternatively you may consider to exploit the fact, that INSR activity is directly connected to its autophosphorylation level. So you may just prepare a cell lysate for western blotting and probe with INSR phospho-specific antibodies. This is a well established indicator for INSR activation and much faster than doing a real in-vitro activity measurement.

In case you would like to do the in-vitro assay I may send you an IP based kinase assay protocol.

Daniel

Thanks Daniel! I used the later approach, but it did show difference between my study groups. I am not sure if it was because of loss of kinase activity or increase in phosphatase activity. May be I will have try the long route!