Harvesting cells for Western - what protease inhibitors do you add to PBS? - (Sep/20/2005 )

Hi everyone,
When harvesting cells for a WB via scrapping....I know most people do it in PBS. I had a question as to what protease inhibitors one needs to add to this PBS and what cocentrations?? I am actually looking at Akt, p-Akt etc at really short treatment periods ie 5 mins, 10 mins. Sometimes my results are not really reproducible. I was wondering if my harvesting in plain PBS might be the reason.

What is your opinion and recipie??
p..

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we successfully use roche's "complete" inhibitor cocktail

mike

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for my protein phosphorylation studies, i scrapp cells washed twice in cold PBS, on ice in lysis buffer containing all protease inhibitors (a complete protease inhibitors cocktail from Sigma) including 0.1 mM of activated orthovanadate

results are reproducible but i do it very rapidly

Seb_

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In your case I'd also recommend using a phosphatasse inhibitor, such as b-glycerophosphate or para-nitro phenyl phosphate.

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But isn't PSB a phosphate buffered saline that will make anti-Tyr P bind everywhere and not to specific protein? You should use TBST instead. Or am I wrong?

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