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Protein dephosphorylation with wich Phosphatase?... and what lysis buffer?


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#1 mirc

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Posted 21 November 2010 - 09:03 AM

Hi there,
I wonder if someone here could help me with this problem:
I have a protein of huge size. Nevertheless I see a shift in mobility in western blot when using a mitotic lysate in contrast to an interphase lysate. I now want to know wether this is due to phosphorylation (I already know from mass spec data that my protein is phosphorylated in mitosis, but nevertheless want to demonstrate it in a different way). So I thought "letīs dephosphorylate with lambda PP". The problem is, as soon as I lyse my cells in our lysis buffer (actually RIPA without SDS) and add no phosphatase inhibitors (Phosstop from Roche) my lysate gets too slimy to pipette or quantify the amount of protein (already tried benzonase treatment - didnīt help).Would you recommend IPing my protein in buffer containing phosphatase inhibitors and then incubate it with the Phosphatase to circumvent this problem?
Furthermore, I once treated the slime with lambda and saw a shift of CDK1 but not on my protein. So what phosphatase do you think is best for treating proteins, since Iīve heard that some proteins are not dephosphorylated by all enzymes...but which to use?
Actually it always sounds so nice and easy in publications "we treated the lysate with PP and voila - the protein shifted down in western" therefore Iīm not sure wether itīs my own stupidity or bad luck...
Thanks for help!
cheers

#2 bob1

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Posted 21 November 2010 - 04:26 PM

The slime is DNA, you can break it by sonication.




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