Immunoaffinity chromatography

Current Protocols in Protein Science
T A Springer

Abstract

This unit describes the isolation of soluble or membrane-bound protein antigens from cells or homogenized tissue by immunoaffinity chromatography. This technique involves the elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. Specifically, antibodies are coupled to Sepharose (an insoluble, large-pore-size chromatographic matrix). High-molecular-weight antigens pass freely into and out of the pores and bind to antibodies covalently bound to the matrix. To elute the bound antigen from the immunoaffinity matrix, the antibody-antigen interaction is destabilized by brief exposure to high- or low-pH buffer. Batch purification of antigens is provided as an alternate procedure that shortens the column loading time. The detergent octyl beta-D-glucoside can be used instead of Triton X-100 for elution. Because octyl beta-D-glucoside has a high critical micelle concentration (CMC), a protocol is provided for its removal by dialysis. The procedure for covalently linking an antibody to Sepharose using the cyanogen bromide activation method is given in a support protocol.

References

Jan 1, 1979·Methods in Enzymology·A HeleniusC Tanford
Jul 1, 1974·Analytical Biochemistry·S C MarchP Cuatrecasas
Dec 22, 1970·Biochimica Et Biophysica Acta·J A van Mourik, I A Mochtar
Jan 1, 1984·Methods in Enzymology·L M Hjelmeland, A Chrambach
Jan 1, 1984·Methods in Enzymology·M WilchekJ Kohn

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