Preparation and application of polyclonal and monoclonal sequence-specific anti-phosphoamino acid antibodies.
This unit discusses the issues that must be considered in the design, production, and characterization of polyclonal and monoclonal sequence-specific anti-phosphoamino acid antibodies. Protocols are provided for generating and purifying such antibodies, and methods are also provided for producing useful polyclonal antibodies in a non-purified form. Support protocols describe coupling of peptides or phosphotyrosine to a solid support for use in affinity chromatography. An example of the generation, purification, and characterization of two sequence-specific anti-phosphopeptide antibodies specific for different sequences of a single phosphoprotein is described. The cross-reactivity of such antibodies, which is a common problem with anti-peptide antibodies, is also discussed.
Sun
Tong
T
University of Texas M.D. Anderson Cancer Center, Houston, Texas.
Arlinghaus
Ralph B
RB
University of Texas M.D. Anderson Cancer Center, Houston, Texas.
D000276:Adjuvants, Immunologic / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000941:Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods; D003432:Cross-Linking Reagents / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000737:chemistry; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D019606:Phosphoamino Acids / Q000235:genetics / Q000276:immunology*; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D000276:Adjuvants, Immunologic; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000941:Antigens; D003432:Cross-Linking Reagents; D017077:Culture Media, Conditioned; D010455:Peptides; D019606:Phosphoamino Acids; D013912:Threonine; D014443:Tyrosine; D012694:Serine