UNIT 9.5 Immunoaffinity Chromatography

  1. Timothy A. Springer

Published Online: 1 MAY 2001

DOI: 10.1002/0471140864.ps0905s03

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Springer, T. A. 2001. Immunoaffinity Chromatography. Current Protocols in Protein Science. 3:9.5:9.5.1–9.5.11.

Author Information

  1. Center for Blood Research Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: MAR 1996


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 β-D-glucoside can be used instead of Triton X-100 for elution. Because octyl β-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.