Production of antibodies that recognize specific tyrosine-phosphorylated peptides.
It is possible to produce anti-phosphopeptide antibodies (i.e., antibodies recognizing phosphorylated peptides) that recognize a protein only in its phosphorylated state, and that do not cross-react with either the cognate unphosphorylated protein or other phosphoproteins. This unit describes production of antibodies against tyrosine-phosphorylated peptides, with which the authors have the most expertise, but the principles discussed here also apply to peptides phosphorylated on serine and threonine. Protocols are presented for the production of polyclonal and monoclonal anti-phosphopeptide antibodies. Support protocols are provided for the coupling of peptides and phosphotyrosine to an affinity matrix (Affi-Gel 10); BSA-agarose affinity matrix is commercially available.
DiGiovanna
Michael P
MP
Yale University School of Medicine, New Haven, Connecticut.
Stern
David F
DF
Yale University School of Medicine, New Haven, Connecticut.
Roussel
Robert R
RR
Dartmouth College, Hanover, New Hampshire.
D000906:Antibodies / Q000276:immunology*; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D010766:Phosphorylation; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; D010748:Phosphopeptides; D014443:Tyrosine; C032392:Affi-Gel 10; D012685:Sepharose