binding studies using receptors isolated from immunoprecipitation - (May/11/2007 )

Hi,
The PPARgamma receptor is expressed in the A549 cells I am working with. I have previously synthesised some compounds which may be potential ligands for this receptor. So to determine this, I would like to do binding assays with my compounds. I could lyse the cells and perform the binding assay directly with the crude lysates, but I was also wondering if it is also possible to isolate the receptor via immunoprecipitation and perform binding studies using the isolated receptor. I have never done immunoprecipitation before, and I do have antibodies for the PPARgamma receptor. Any feed back would be greatly appreciated.

Thanks

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receptors may lose some binding capabilities after IP or even after lysis; why not directly working with cells? how do you monitor the binding activity?

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Thanks for the reply. I plan to do competitive binding experiments using a tritiated ligand for the receptor. Will then see if my compounds can displace the tritiated ligand by measuring any decreases in radioactivity. The reason why I wound prefer to work with purified receptor is so that I can determine if my compounds are binding specifically to the PPARgamma receptor. If perform binding in cell or crude cell lysate, then I can't be certain if any binding detected is PPARgamma specific.

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