2 replies to this topic

[Chris_NL]

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?

Cheers,

Chris

[bob1]

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.

[knuf]

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