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IHC: confirming specificity phosphorylated protein antibody

IHC

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#1 Chris_NL

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Posted 08 July 2012 - 11:25 PM

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

#2 bob1

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Posted 09 July 2012 - 12:55 AM

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.

#3 knuf

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Posted 13 July 2012 - 06:14 AM

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





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