How to the modification of a protein? - (Feb/25/2007 )

Hi, every one:
I am studying a protein in a signaling pathway. recently, I use the Ab of this protein to detect the change in my treated samples and found that besides the band of the expected size of the protein, there is another shifted band right above the band. I suspect that the upper band could be the modified version of the protein: either phosphorylation of mono-ubq. I think it is more possible to be the phosphorylated protein.So my questions are:
1. how to determine the upper band presents the modification of the protein?
2.As no one so far has reported phosphorylation of the protein and of course no Ab available to detect its phosphorylation, so how to test whether the upper band is phosphorylation of the protein?

Thank you very much.

1. how to determine the upper band presents the modification of the protein?

mass spec would be a good way.
the other way is to treat with a phosphatase and see if you can recover the original band, and then perhaps put it back into the phosphorylated form with a non-specific kinase.

2.As no one so far has reported phosphorylation of the protein and of course no Ab available to detect its phosphorylation, so how to test whether the upper band is phosphorylation of the protein?

try anti-phosphorylated amino acid residue antibodies - anti-pTyr, anti-pSer, anti-pThr; they usually work most of the times.


-AA, Salk/UCSD Biology Grad student