Phosphatase treatment as a control for Phospho antibodies - (Aug/09/2008 )
HI,
In western blots, a common negative control for phospho antibodies consists of pre-treating the tissue with phosphatase. If the antibody is phospho specific (for whatever protein, let's say P-AKT or P-EGFR), then the signal should go away after phosphatase treatment. Usually gama phosphatase or calf alkaline phosphatase is used.
I am wondering why I don't see this technique used more often in IHC. I would like to verify the phospho specificity of an IGF1R-P antibody that I am using right now for IHC, but I can't seem to find a protocol for IHC pre-treatment with phosphatase.
Any thoughts would be greatly appreciated.
-Aettius-
QUOTE (Aettius @ Aug 9 2008, 06:28 AM)
HI,
In western blots, a common negative control for phospho antibodies consists of pre-treating the tissue with phosphatase. If the antibody is phospho specific (for whatever protein, let's say P-AKT or P-EGFR), then the signal should go away after phosphatase treatment. Usually gama phosphatase or calf alkaline phosphatase is used.
I am wondering why I don't see this technique used more often in IHC. I would like to verify the phospho specificity of an IGF1R-P antibody that I am using right now for IHC, but I can't seem to find a protocol for IHC pre-treatment with phosphatase.
Any thoughts would be greatly appreciated.
In western blots, a common negative control for phospho antibodies consists of pre-treating the tissue with phosphatase. If the antibody is phospho specific (for whatever protein, let's say P-AKT or P-EGFR), then the signal should go away after phosphatase treatment. Usually gama phosphatase or calf alkaline phosphatase is used.
I am wondering why I don't see this technique used more often in IHC. I would like to verify the phospho specificity of an IGF1R-P antibody that I am using right now for IHC, but I can't seem to find a protocol for IHC pre-treatment with phosphatase.
Any thoughts would be greatly appreciated.
after which step do you like to dephosphorylate? fixation and at least partial denaturizing of proteins reduce availability of phosphoproteins for phosphatases...
-The Bearer-