You might try cation-exchange of your protein, depending on its pI and
sensitivity to pH. Protein phosphorylation sites are generally accessible not
only to a kinase but also to a chromatographic stationary phase surface. This
makes them good potential contact regions. On pg. 3 of our current brochure
is an example of phosphorylation variants of H1 histone from Tetrahymena, with
anywhere from 1-7 phosphates. The monophosphorylated form(s) elute about 10
minutes (@ 5 peak widths) earlier than the nonphosphorylated form, since the
phosphate group repels the negatively-charged cation-exchanger.
RE: Ken Mitchelhill's comments: Since chromatography involves surface
contact, it can distinguish between variants not only on the basis of how many
phosphates the protein has but also where they are on the surface of the
tertiary structure. The above chromatogram has two peaks with 1 phosphate,
two with 2, at least two with 5, and two or three with 6. There seems to be a
preferred sequence in the phosphorylation of these sites, judging from the
ratio of the variants. Thus, per Ken's comments, you may find 90% of the
protein is not phosphorylated, 5% has 1 phosphate, and 5% has 3-5 phosphates
per protein.
Andy Alpert
PolyLC Inc.
9151 Rumsey Road, ste. 180
Columbia, MD 21045
tel: 410-992-5400 FAX: 410-730-8340 e-mail: PolyLC@aol.com