Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4303

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Immunoaffinity Purification: Coupling Antibodies to Protein A or G Bead Columns

Ed Harlow and David Lane

This protocol was adapted from "Immunoaffinity Purification," Chapter 9, in Using Antibodies by Ed Harlow and David Lane. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1999.

The first 100 words of the full text of this article appear below.


INTRODUCTION

Protein A/protein G bead-antibody columns are one of the most versatile column matrices used for affinity purification of antigens. The columns are easy to prepare, and because the antibody molecules are bound to the matrix via the Fc domain, the antigen-binding site is correctly oriented for maximal interaction with the antigens. In the approach described here, antibodies are bound to protein A or protein G beads and are then crosslinked to the matrix via a bifunctional coupling reagent called dimethyl pimelimidate (DMP). Columns are ready for antigen binding (see Immunoaffinity Purification: Binding of Antigen to Antibody-Bead Matrix in a Column . . . [Full Text of this Article]


MATERIALS

Reagents

Equipment


METHOD


TROUBLESHOOTING


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