Protein Protocols Protein Protocols

Immunoblotting Using Secondary Ligands
54
Detection of Polypeptides on Immunoblots Using Enzyme-
Conjugated or Radiolabeled Secondary Ligands
Nicholas J. Kruger
1. Introduction
Immunoblotting provides a simple and effective method for identifying specific
antigens in a complex mixture of proteins. Initially, the constituent polypeptides are
separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE), or a similar technique, and are then transferred either electrophoretically or
by diffusion onto a nitrocellulose filter. Once immobilized on a sheet of nitrocellulose,
specific polypeptides can be identified using antibodies that bind to antigens
retained on the filter and subsequent visualization of the resulting antibody-antigen
complex. This chapter describes conditions suitable for binding antibodies to immobilized
proteins and methods for locating these antibody-antigen complexes using appropriately
labeled ligands. These methods are based on those of Blake et al. (1), Burnette
(2) and Towbin et al. (3).
Although there are several different techniques for visualizing antibodies bound to
nitrocellulose, most exploit only two different types of ligand. One is protein A conjugated
to a marker enzyme, radiolabeled or otherwise tagged. The other ligand is an antibody
raised against immunoglobulin G (IgG) from the species used to generate the primary
antibody. Usually, this secondary antibody is either conjugated to a marker enzyme or
linked to biotin. In the latter instance, the biotinylated antibody is subsequently
detected using avidin (or streptavidin) linked to a marker enzyme.
Detection systems based on protein A are both convenient and sensitive. Protein A,
from the cell wall of Staphylococcus aureus, specifically binds the Fc region of IgG
from many mammals (4). Thus, this compound provides a general reagent for detecting
antibodies from several sources. Using this ligand, as little as 0.1 ng of protein may be
detected, although the precise amount will vary with the specific antibody titer (5). The
principal disadvantage of protein A is that it fails to bind effectively to major IgG
subclasses from several experimentally important sources, such as rat, mouse, goat,
and sheep (see Table 1). For antibodies raised in such animals a similar method using
derivatives of other bacterial immunoglobulin-binding proteins may be suitable
(see Note 1). Alternatively, antibody bound to the nitrocellulose filter may be detected
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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