Antibodies to phosphorylated amino acids

Richard Miller rmiller at bimcore.emory.edu
Fri Jan 27 01:58:42 EST 1995


The use of  antibodies against phosphorylated amino acids depends
principally on the phosphorylated amino acid in question.  It seems that the
anti-phosphotyrosine antibody works well, since the antigenic group is fairly
well removed from the peptide backbone.  We've been trying to use anti-
phosphoserine antibodies to identify peptide fragments which we know contain
a phosphoserine (through proteolytic digestion of radiolabelled cells followed
by TLC and autoradiography).  We have thus far failed to achieve a positive
response from our peptide as well as from a number of positive controls
(including egg white riboflavin binding protein which contains no less than
seven phosphoserine residues).  The product literature from the companies
supplying these antibodies indicates that the immunogen was a phosphorylated
serine containing peptide.  The anitibody is said to recognize phosphorylated
peptide over non-phosphorylated peptide and p-Y and p-T containing peptides.
I feel that the response observed is these assays is more dependent on the
particular amino acids surrounding the p-S than the p-S alone.  As p-T is not
really that much more distinct an anitigenic group than p-S, I imagine these
antibodies are probably not particularly good either.  If you know you have
a p-Y residue, I think Westerns are a good choice, but I'm still not convinced
that anti-PS and probably anti-PT are all that useful.

Richard Miller
Graduate Student
Department of Biochemistry
Emory University



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