tips for detect phosphorylated proteins - (Jul/26/2007 )

Hi, there,

recently I tried to do western to detect phosphorylated proteins, but it never worked. Everytime there is a high-molecular weight band (250kD)in each lane. I don't know what is wrong. When I reprobe with other antibody, like non-phosphorylated or actin, they woked well. Does anybody have any idea about it? thanks a lot.

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tips.....
well use 4%BSA in TBST for blocking
Don't use tween in 1st AB ans washes
use TBST with your 2nd ab.
Transfert gently and use NaF in all solutions of AB (as recommended by santa cruz)
1st AB ovenight 4°C and second 1h at least (but not too much!)
don't check by ponceau or coomassie the transfert. better is to stain the gel

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I can only state some advices: as fred_33 suggested use phosphatse inhibitors in your lysis buffer, f.i. PhosSTOP of Roche;

you need an external reference; some suppliers provide qualified lysates ( f.i. Upstate) to detect certain antigenes


specific phosphorylations depend on status, stress or stimulation of cells; may be you do not meet these conditions to find certain phosphorylations

you ~250 kDa signal may be an aggregate of several (phospho-)proteins; reduce heating of sample, use 65°C or 37°C to prepare for gel running

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