Working with Phospho-antibodies in Tissues - Phospho antiboedies (Mar/27/2007 )

Hi everyone,

I'm trying to use phospho S33/37/Thr41 and S45 beta-catenin and non-phospho beta-catenin antidodies with tissues. Non-phospho antibodies work well, but the others not. Phospho-antibodies work in SW480 cells with weak signals that has a lot of beta-catenin protein. I think I should load more protein to see phosphoylated form, shouldn't I ? Is there anybody work with using phospho antibodies in tissues, I'm not sure that are work in tissues ? Thanks a lot.

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what do you mean by "load more protein"? Which protein?

Phosphosignals are in most cases dynamic, and reflects distinct situations of a cell which means you will find specific phosphorylations not at any time and any condition

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Hi,

Phosphorylation of the beta-catenin sign the protein for ubiqutin degradation. I want to see phosphorylation status for these aminoacids in tumor and normal tissues.

I mean load more total protein too see phospho S45 and S33/37/Thr41 beta-catenin. Phospho form of the protein is less than total beta-catenin, so I think I can rise the antigen. Isn't this work?

Thanks a lot again.

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but how will you load more protein in a tissue??? you mean by overexpression??

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Hi Bearer,

I mean load more tissue lysate, eg. loading 70ug protein instead of 30ug protein to rise antigen on membrane.

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You could try that.

But, if the phosphorylation of the protein leads to its degradation, you should not see a lot of phosphorylated protein. So, you could try to load more proteins, or block the proteasome and then blot the phospho-protein.

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ok, then I was puzzled by the topic headlline, "phospho-antibodies in tissues" imply immunohistochemistry... but you think of immunoblotting;

are you working with mini, midi or large gels?; for mini gels, 70 µg per lane will definitely be overloaded

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I'm working with mini gels, I know it will be overloaded but I want to rise phospho antigen. I did not see bad results. I saw phospho signals on cell line lysates but not in tissue lysates.

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instead of increasing total amount protein, rather use a phosphatase inhibitor cocktail to save phosphorylation during tissue lysis

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Hi, I'm using okadaic acid and beta-glycerophosphate as phosphatase inhibitors.

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