I have been trying western blot to estimate p-IR/IGFR-1 (not abundant in brain) but its not the best technique in my case. I am looking at very specific regions in the brain and as a result I collect tiny amounts of protein from tissue lysates. Its not feasible for me to load 40-50 ug protein in the SDS PAGE gels. No luck with different antibodies. Has someone used any other molecular technique to look at p-IR/IGFR1? Any suggestions?
Try immunoprecipitation with phosphotyrosine antibodies conjugated to agarose beads and then blot with anti Insulin/IGFR antibody. This works fine.
If tissue amount is very small such that the conventional Phosphotyrosine enriched proteins do not give you adequate specific signal, you have two options: combine samples from multiple animals; or to increase sensitivity of detection, use P32 loading of animal. Not sure that the latter will be possible but in isolated mitochondria, I have successfully done it.
Immunoprecipitation with anti-IR/IGFR, followed by western blot by p-Tyr antibody. This should work.
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