I am running a immunohistochemistry (IHC) for a specific phospho-protein and my PI asked for a control that should be the same tissue treated with phosphatase prior the staining just to confirm if the antibody is binding to a phospho-protein. I ran this IHC control twice with different phosphatase treatments (rSAP and lambda) and I got no difference at all in comparison with no phosphatase treated slides. At this point, I am wondering if I can really dephosphorylate proteins in an already fixed tissue. If anybody have done this in the past, please let me know the protocol you did.
I had worked with antibodies (to the same molecule) anti phospo-protein and other domain without phosphate in the same tissue to show activation status. I had never heard about dephosphorylation after fixation process, but it is not a impossible. What sometimes occurs is that some proteins loses detection after cutting even when slides are frozen preserved (FFPE slides). Maybe you can generate a control with tissue or culture cells pre treated with phospathase.
Thank you for spending time to help me. Natalie Tulchin , I am not using a kit, I just tried to incubate the slides using NEB rSAP and lambda PP following NEB's instructions for regular dephosphorylation reactions, I just changed the time of incubation (overnight for rSAP and 4h for lambda PP). Actually, after a second look on the slide treated with lambda phosphatase I feel I have a weaker staining when comparing to control not treated with lamda PP.
Having achieved a partial dephosphorylation by prolonging the treatment of the target tissue with lambda phosphatase, you may get a better result by sequentially increasing the treatment time.
- Badges
- Science topic
- Similar topics
- Biological Science
- Kinesiology
- Running