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  Home > JCE Print > Journal of Chemical Education > Issues > 2000  > November  >
In the Laboratory
Immunoprecipitation of Serum Albumin with Protein A-Sepharose: A Biochemistry Laboratory Experiment
Robert C. Bohinski
Department of Chemistry, John Carroll University, University Heights, OH 44118
Cover
November 2000
Vol. 77 No. 11
p. 1460
Abstract
An exercise has been designed and optimized to acquaint students with the simple yet powerful technique of immunoprecipitation. Protein A-Sepharose (PA-S) is used as a solid-phase precipitant to recover bovine serum albumin (BSA, the antigen) recognized by anti-BSA antibody (Ab). The high degree of binding specificity between antigen and antibody is illustrated by recovery of BSA from a complex mixture of proteins obtained from wheat germ and chicken breast. Various controls are included for a thorough data analysis. The solid phase of Ag/Ab/PA-S is recovered by centrifugation, thoroughly washed, and treated to dissociate the BSA antigen. Samples are examined by discontinuous denaturing gel electrophoresis (SDS-PAGE) with Coomassie blue staining. The supernatants, containing proteins that are not precipitated, are also analyzed. Antigenic cross-reactivity, ranging from strong to none, is demonstrated in a second part by using serum albumins from seven different sources. Systems can be set up, shaken, and prepared for electrophoresis in a single lab period with time for laboratory lecture and discussion about antibody structure and function, antibody-based methods in general, and immunoprecipitation in particular.
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*  Citation
 Bohinski, Robert C. J. Chem. Educ. 2000 77 1460.
*  Keywords
 Laboratory Instruction; Biochemistry; Proteins / Peptides; Electrophoresis; Separation Science
*  History
 Created:
Last Updated:		 October 6, 2000
August 31, 2005
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