Phosphatase treatment

Phosphatase treatment

Postby nazrul » Fri Aug 19, 2011 5:56 pm

Hi Guys
I have just registered in this forum and looking for some one who can provide me with some tips for my experiment. Using Pro-Q diamond staining, I found some protein spots in my 2DE gels that are phosphorylated. Now I am trying to confirm them by using phospphatase. Does anyone have a protocol on how to treat a protein sample in 8M urea, DTT, CHAPS with phosphatase?
naz
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Re: phsophstase treatment

Postby cicenasj » Sat Aug 20, 2011 12:10 am

the best is to dialize your sample against milipore H2O. Then incubate dialyzed proteins 30 min. at 30C 9 u/µl lambda protein phosphatase (NEB) in this buffer:

50 mM Tris-HCl, pH 7.5
0.1 mM EDTA
2 mM MnCl2
5 mM DTT

Do not forget control with only buffer (no phosphatase)!

Good luck!
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Re: Phosphatase treatment

Postby DNAer » Fri Feb 15, 2013 1:10 pm

nazrul wrote:Hi Guys
I have just registered in this forum and looking for some one who can provide me with some tips for my experiment. Using Pro-Q diamond staining, I found some protein spots in my 2DE gels that are phosphorylated. Now I am trying to confirm them by using phospphatase. Does anyone have a protocol on how to treat a protein sample in 8M urea, DTT, CHAPS with phosphatase?
naz


phosphotase will be inhibited by 8M urea, definitely!
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