Hi,
I want to test GSK-3b activity in my cells so I thought I would do a western blot on total cell lysate, for b-catenin (hoping increased GSK activity will show as decreased b-catenin), I have noticed in papers they mostly blot for the p-b-catenin, or total b-catenin in nuclear extracts, however I have had problems with phospho antibodies beofre, and my lysate is alreay total ccell lysate. I have only found one paper did this and they didn't get such a great result (the image was a little hazy). Does anyone know if this is ok to do and a valid way to measure GSK activity, total b-cat in total cell lysate????
Thanks!
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#2
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Posted 09 June 2009 - 09:27 AM
Why not blot with phospho-Gsk3? Phosphorylated protein is not active! I would conjugate those two results, phospho-gsk3 and total cytoplasmic b-catenin.
Or IP b-catenin and blot for ubiquitination. Or block proteasomal degradation with both/either MG132 of lactacystin and blot for b-catenin. There are a lot of things you can do
Or IP b-catenin and blot for ubiquitination. Or block proteasomal degradation with both/either MG132 of lactacystin and blot for b-catenin. There are a lot of things you can do
#3
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Posted 10 June 2009 - 02:27 PM
Hi! Thanks for the reply, very helpful!
I am going to blot with p-GSK too, but so far I havn't had much luck so am still optimising it. I dont think we have either of the proteasomal degradation blocking agents already in the lab but will look into those. But I'll order the b-catenin and then can go ahead with the p-GSK and b-catenin blots.
Thanks again!
I am going to blot with p-GSK too, but so far I havn't had much luck so am still optimising it. I dont think we have either of the proteasomal degradation blocking agents already in the lab but will look into those. But I'll order the b-catenin and then can go ahead with the p-GSK and b-catenin blots.
Thanks again!
Edited by scistudent, 10 June 2009 - 02:30 PM.
