phospho akt and akt - does p-akt antibody recognize normal akt? (May/19/2011 )

hi guys,

i probed my blot with phospho akt antibody... then i stripped and probed with general akt antibody.... amazingly, i see the same bands. my question is does normal akt antibody recognize phospho-akt? how do you guys probe for p-akt and normal akt?

thanks a bunch in advance...

SHOULD the bands for p-akt and akt look different?

AKT total ofcourse shd recognise the whole AKT expressed, but your pAKT is specific to the phosphorylated form alone, the difference you shd see is if you run the samples with proper quantification, you shd see the Normalised AKT total and ups and downs in your pAKT. in your blot it might be the case where your pAKT is not altered in any of ur samples and thus may look the same like AKT total or between themself.., or it could also because of improper stripping that still keeps your previous pAKT....

you shd see the band for AKT and pAKT resolved almost at the same position in this case you dnt see much difference...

Gnana...,

GNANA on Thu May 19 13:26:34 2011 said:

Total Akt definitely recognize the phospho Akt.

If you have got the identical bands for both, so probably you should check that you have done stripping correctly or not by exposing the blots to secondary antibody after stripping, if you still see the same size of bands as per the exact previous positions means stripping is improper. Usually, stripping for 15 min.s at room temperature on shaker is sufficient enough to remove phosphorylation of proteins.