UNIT 13.6 Preparation and Application of Polyclonal and Monoclonal Sequence-Specific Anti-Phosphoamino Acid Antibodies
Published Online: 1 FEB 2004
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Protein Science
How to Cite
Sun, T. and Arlinghaus, R. B. 2004. Preparation and Application of Polyclonal and Monoclonal Sequence-Specific Anti-Phosphoamino Acid Antibodies. Current Protocols in Protein Science. 34:13.6:13.6.1–13.6.27.
- Published Online: 1 FEB 2004
- Published Print: NOV 2003
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.