western blot for phosphorylated proteins - (Aug/03/2007 )

Hi,

I am not sure about the right forum for this question. I am sorry if I am in the wrong side.

I want to do western blot for some phosphorylated proteins like p-c-Jun, p-PERK, p-Akt, p-eIF2, p-IRS1 to study the ER stress in a mice model. Any one who is currently doing this could help me for my succesful WB.

My questions are:

Should I use any phorylation inhibitors?
Should I use any special techniques for sample preparation?
Is there any special tips?

Please post your suggestions

EKG

Hi! Not sure if what I do is 100% correct, but what I do is supplement my RIPA (or other lysis) buffer with a Ser/Thr phosphatase inhibitor cocktail from Sigma, add some activated sodium vanadate stock (I've read this inhibits Tyr phosphatases), in addition, I always add PMSF, and the Roche complete protease inhibitor cocktail. Others around here add beta-glycerol-phosphate as a P-tase inhibitor.

I work on ice when scraping cells. I always use 5% BSA in TBST to block my membranes instead of 5% milk, because I've read that casein is highly phosphorylated and will make your western blots more dirty from non-specific binding of the Ab to p-casein.

Hope this helps a bit!

phosphatase inhibitors in all extraction procedures. when you start at collecting cells, work at 4° or on ice.
SDS page slow 100V and maybe at 4° too.
BSA for blocking agent and no milk
phosphatase inhibitor in all western steps
no stripping possibility

Hi EKG,

Just wonderign what P-tase inhibitors you use? this would get very expensive if I had to use my purchased cocktail from Sigma....

Do you include the P-tase inhibitor during the 1' Ab incubation as well?

many thanks!