phosphorylated protein Western blotting - HELP!! (Jul/13/2006 )

I'm trying to detect phosphorylated Rb protein from mice ovary.

i used protease inhibitor on the lysis buffer.

But i couldn't detect ANY signal on film.

Do i need to use 'phosphatase inhibitor' in my buffer?

Is that nessesary? Can it be the problem of that result?

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Are you using TBS for your blocking, diluting Ab and so on? Proteases inhinitors should be used to avoid proteases action in your extract

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I would add some phosphatase inhibitors to your buffer like sodium orthovanadate or sodium fluoride and include a postive control for Rb phosphorylation if you do it again next time

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what do you mean by ANY signal?
you can't see proteins, or you can't see phosphorylated proteins?

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This is exactly why I'm asking if he/she uses TBS. If he/she uses PBS, it could be that the Ab doesn't bind the phospho-protein because of the eccess of phosphate in the PBS. In that case it could see he/she doesn't see any signal on the membrane using anti-phospho-Rb. If this is not the case, it could be there's no proteins on the membrane...

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^^

yes i'm using TBST buffer for washing, and dilution of Abs.

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i check the protein transfer by ponchu.
And transfer was well done.

but i i couldn't detect the phosphorylated protein on film after ECL detection.

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Are you sure your Ab works? Can you try it on samples you know contain the phosphorilated protein FOR SURE?

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can you detect the Rb protein on your blot? (I'm not talking about phosphorylated Rb)

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hi,
i wud look for total RD protein as suggested by MISSILE, have a phosphorylated protein control on ur western blot. then compare both signals.
r u really taking care of ur samples, processing samples on ice, immediately adding lamile buffer and so on....
because in my experience with the time and raise in temparature, proteins get dephosphorylated.

regards
payeli.

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