standard 2-D electrophoresis generally resolves phospho- from
dephopho-proteins. In my experience, most conventional liquid
chromatography methods don't have sufficient resolution to disriminate a
monophosphorylated protein from it's non-phosphorylated counterpart
although if the phosphoprotein is poly-phosphorylated it may (?) separate
on ion-exchange at acid pH. Nice thing about 2DE is that it can give you an
idea of the number of phosphates and relative abundance of each species.
A word of warning, don't assume 30% 32P incorporation means "stoichiometry
of about 0.3", I've got a shocker at the moment with apparent 40% 32P
incorporation but 2DE shows a train of 4-6 spots (depending on the
imagination) and the old "gold standard" Hunter et. al. phosphopeptide maps
show at least six labelled peptides, maybe more.
Good luck....Ken
>What is the best method for seperation of a phospho-protein from its
>dephospho-protein counterpart following in incomplete in vitro
>phosphorylation reaction (stoichiometry of about 0.3). My septigenerian
>boss suggested a Mercury column. I am trying an Fe3+-IDA (metal chelating
>column). This column has been shown to selectivly bind phosho-amino acids
>at lower pH. It isn't working for my protein. Are there other methods of
>seperation of prepartive amounts of phospho-protein? Thank you imensly.
>
>James Bibb
>c/o Joe Fernandez
>
>Joseph Fernandez
>Associate Director
>The Rockefeller university
>Protein/DNA Technology Center
>1230 York Ave. New York, NY 10021
>Phone: (212)-327-8869
>FAX : (212)-327-8620
>email: fernaj@rockvax.rockefeller.edu
>Lab Web Page: http:\\pdtc.rockefeller.edu
********************************
Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA
Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676
Email: k.mitchelhill@medicine.unimelb.edu.au
Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.abrf.org
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