I would like to check in vivo insulin action, by assessing the IRS-1 and phospho-IRS-1, together with Akt and phospho Akt in adipose tissue after insulin administration. Any suggestions on protocols, and antibodies to purchase are welcome!
Is that a gerneral question on the perfect western blotting antibodies or do you need some advices for determining these specific proteins?
I would like to do Western blots of these proteins (Phosph-IRS-1 and phospho-Akt), so I would like to know if anyone has used these antibodies for Western blots. The general thing was about the "in vivo insulin action" protocols.
Then I got you right. In that case, I can't help you. But certainly others. Wish you good luck.
you can check in my previous publication if you need (Corporeau)
Dear Charlotte, which one of your publications?
Dear Santosh, thanks for the heads up!
Sinem,
I have performed similar experiments and have some recommendations. For AKT and pAKT (S473), I would highly recommend purchasing from Cell Signaling Technologies. In addition, the pIR/pIGFR from this vendor is also excellent. IRS-1 phosphorylation is a little more difficult as the "gold standard" use to be to check tyrosine 608/612 phosphorylation (PI 3-kinase binding site) using phospho-specific antibodies, but the only good antibody that I have found is sold by Millipore and is no longer available. I am currently screening other commercially available antibodies and will make sure to post any positive results that I obtain. One alternative is to perform anti-IRS-1 IP's and subsequently blotting with anti-phosphotyrosine antibodies. Good luck with your experiments.
Hi Sinem,
I'm looking to buy an antibody against phospho-IRS-1 too. Did you have any luck figuring out which one works?
Thanks!
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