Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4304

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Immunoaffinity Purification: Coupling Antibodies to Activated Beads

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

Purified antibodies are bound to commercially available beads that have been activated using a chemical reagent. Many of the coupling methods yield a linkage that is stable to a wide range of denaturing conditions. Antibody-bead complexes are ready for antigen binding (see Immunoaffinity Purification: Binding of Antigen to Antibody-Bead Matrix in a Column or Immunoaffinity Purification: Binding of Antigen to Antibody-Bead Matrix in a Suspension) and subsequent elution (Immunoaffinity Purification: Eluting Antigens from Immunoaffinity Columns).


MATERIALS

Reagents

Activated beads


Coupling mechanisms commonly used in commercially available activated beads.

Coupling mechanism Binding group on protein Stability of final linkage
Table 1 1,1'-Carbonyldiimidazole -NH2 Avoid pH >10
Table 1 Cyanogen bromide -NH2 Good, some leaching
Table 1N-Hydroxysuccinimide -NH2 Excellent
Iodoacetyl -SH2 Excellent
Table 1 Tresyl chloride -NH2, -SH2 Excellent

caution Ethanolamine (100 mM, pH 7.5)

caution Merthiolate

NaCl (1 M) with sodium . . . [Full Text of this Article]

Equipment


METHOD


TROUBLESHOOTING


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