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.