Protocol

Directed Orientation of Antibodies during Preparation of Antibody Affinity Resin

This protocol was adapted from "Affinity and Immunoaffinity Chromatography," contributed by Keith Brocklehurst, Albert J. Courey, Sheraz Gul, Sue-Hwa Lin, and Robert L. Moritz, Chapter 10, in Purifying Proteins for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004.

The first 15% of the full text of this article appears below.

INTRODUCTION

The conventional method of immobilizing antibodies on solid matrices, using cyanogen-bromide-activated Sepharose, often generates affinity columns with low antibody activity because the immunoglobulin molecules are oriented randomly and attached at multiple sites to the Sepharose, which reduces the efficiency of antibody/antigen interactions. This protocol describes the directed coupling of antibody to protein G-agarose via its Fc domain, and subsequent covalent cross-linking of the complex using dimethyl pimelimidate (DMP). Protein G binds to the Fc portion of immunoglobulin molecules, allowing optimal spatial orientation of antibodies and maximum antigen-binding efficiency.

MATERIALS

Reagents

Antibodies . . . [Full Text of this Article]

Equipment

METHOD