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