immunoprecipitation question - (Nov/08/2006 )
hi guys,
i have a cell line which expresses abundant insulin receptor, and i want to measure how active [i.e. phosphorylated] it is under certain conditions. i have been told i can do this via immunoprecipitation using an anti-phospho tyr antibody. can anyone tell me what i need (i.e. immunoprecipitation protein a/g beads etc) and what a good protocol would be?
regards
i have a cell line which expresses abundant insulin receptor, and i want to measure how active [i.e. phosphorylated] it is under certain conditions. i have been told i can do this via immunoprecipitation using an anti-phospho tyr antibody. can anyone tell me what i need (i.e. immunoprecipitation protein a/g beads etc) and what a good protocol would be?
regards
a good provider for antibodies also provides specific protocols for Wb, IP or ICC/IHC of his Ab; for instance, if you try Cell Signalling they provide besides anti-PY Ab a agarose-beads labelled anti-PY Ab; we recently used this antibody to enrich PY-proteins by IP; in this case you will not need protein A or G; along with the Ab you get a detailed instruction which is also to download; in your case, application of IP will enrich IGF-R; as I understand your cell line overexpresses IGF-R you may succeed by direct application of lysats to SDS-PAGE and Wb with a Wb-compatible anti-PY Ab, so there may be no need for specific enrichment by IP
either you can find a antibody directed against the phosphorylated form of the receptor, and an antibody that recognize the receptor independently of its phosphorylaiton state, and then you can directly blot a total extract with the anti-phospho insulin receptor, then strip and then blot with anti-insulin receptor to show that you loaded an equal amount.
or, you don't have an antibody directed against the phosphorylated form :
you would need to immunoprecipitate.
either you precipitate all phosphorylated proteins, and blot with anti-insulin receptor (method 1),
or you immunoprecipitate the insulin receptor, blot with an anti phiospho proteins , like 4G10, that recognize any phosphorylated proteins, and then you strip, and blot again with anti-insulin receptor to show that you loaded the same amount (method 2)
I don't recommend you the method one. It seems faster, but you are not able to demonstrate clearly that you increase the phosphorylation of the receptor with this method.
You could have an increase of the phosphorylation, but it could also be due to an increase of the expression of the receptor, so you would also see more phoshorylated receptors, even if the ratio active versus inactive is still the same.
with method 2 you would see if you still have the same amount of receptor, and you can really conclude that your treatement increase the phosphorylation of the receptor.
before I give you protocols, tell me what method you want to do.
check carefully if you can find an antibody directed against the phosphorylated form of the receptor. Now there are more and more anti phospho antibodies.
this would be the easiest way.
hi and thanks for the swift replies,
i have already ordered some protein A/G beads and an anti-phospho tyr antibody. something like the seconf method you mentioned is probably what i'd do missele
regards
james
OK,
this is the protocol I follow, but I lyse my cells in triton X-100, but i don't do a pre-clearing, but I pre-incubate the protein G in the buffer containing 3% BSA for 1 hour.
thank you very much!