protein an its phospho-protein in the same membrane - (Jan/24/2008 )

I want to do a western of a protein and its phospho-protein using the same membrane. I have seen that people do it, someone knows how? The protein is Akt.

Thanks!!---

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What we are doing is just to blot Akt after blotting of pAkt. We only wash the blotted membrane with TBST for 20min.

If the signal of phospho-protein is a little bit strong, I was told to strip the membrane first. But I didn't try that so far.

-Twisters-

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Yeah thats pretty much the same as i do

though i wash in stripping buffer then in PBS-T before blocking and incubating overnight with total Akt antibody (i use the ones from cel signalling technology)

after developing total Akt i inactivate the hrp using ponceau for 10 min before washing and probing with beta actin (loading control)

cheers

j

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to avoid stripping, use Ab´s of different species subsequently, f.i. mouse anti-Akt, and rabbit anti-phospho-Akt

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