IHC: confirming specificity phosphorylated protein antibody - (Jul/09/2012 )
I want to do stainings on FFPE tissues using an antibody specific for a phosphorylated protein. Now as a control I want to show that this antibody really only binds to the phosphorylated protein. I've heard this can be checked by using a phophatase treatment, after which the staining should be negative. But I'm having a hard time finding a protocol, could anyone help me out?
You should just be able to put some alkaline phosphatase on the slides and incubate at room temp for about 30 min. You can also do a peptide competition where you pre-incubate the antibody with the specific peptide and then use the antibody for detection - the loss of signal indicates specificity.
Preferably you would also determine that the antibody has a single band on a western blot - which is a good indication of specificity.
Just as an addition to what Bob said (which are all good suggestions) with the peptide competition assay, you should see decreased reactivity when pre-incubated with the phospho-peptide but not with the unphosphorylated peptide