Immunoprecipitation: Variations, Considerations, and Applications.

ABSTRACT

Immunoprecipitation (IP) refers to methods of affinity chromatography that enrich and/or purify a specific protein from a complex mixture using a specific antibody immobilized on a solid support. Several operations and processes that are dependent on the isolation, concentration, and modification of proteins have seen improvement in their selectivity and separation based on the integration of IP-specific reactions into their workflows. This relatively simple principle has contributed significantly to our understanding of proteins and their behaviors and has become increasingly fundamental to most protein characterization studies today. In this chapter, we review the basic principles of IP and the several factors that influence each stage, and subsequently the success, of an IP experiment. Moreover, variations in application of the IP principle are discussed, and the adaptability of the techniques based on such is highlighted in the provision of two IP workflows to purify a particular protein from an entire cellular proteosome. These workflows cover the preparation and fractionation of crude cellular lysate into individual subcellular fractions, through to both "batch" and "column"-based extractions of the target protein of interest. Protocols for determining the validity of the workflows, and the presence/abundance of the protein of interest, are also briefly described.