Hi Lisa: I have used a three with a lot of success. There are also several
reports in the literature. Generally, the antibody mix as a polyclonal is
about 5 to 10 against the phosphopeptide and the rest against the peptide.
Works best for a specific domain that contains the phosphorylation site.
peptides longer than 15 residues work better than shorter ones. Clean the
polyclonal first over a P-Thr/P-Ser/P-Tyr-agarose. Take the bound fraction
and run it over a dephospho column to remove all traces of
non-phosphospecific IgGs. The final fraction should be highly specific for
the phospho-domain (epitope). I think one of the one's we made here is
being tested and I am told that it is verey specific for the
phospho-enzyme. gautam
Gautam Sarath
N-226, Beadle Center
Protein Core Facility - Center for Biotechnology &
Department of Biochemistry
University of Nebraska-Lincoln
Lincoln, NE 68588-0664
Phone: 402-472-2928
FAX: 402-472-7842
http://www.biotech.unl.edu/Proteins/index.html