Analysis of Protein Expression in Brain Tissue by ELISA - Drugs of Abuse: Neurological Reviews and Protocols - page 277

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Analysis of Protein Expression
in Brain Tissue by ELISA
Steffany A. L. Bennett and David C. S. Roberts
1. Introduction
The enzyme-linked immunosorbent assay (ELISA) technique offers a sensi-
tive, simple, and versatile method for quantifying as little as 100 pg of target
protein in mixed cell or tissue lysates. A further advantage to the protocol
is the ability to process rapidly and reproducibly large numbers of samples
with minimal equipment requirements. The underlying principle depends on
formation of an antigen-antibody complex immobilized on plastic microtitre
plates. Figure 1 illustrates the basic methodology. In the antibody-sandwich
ELISA, a primary antibody directed against a protein of interest is bound
(adsorbed) to the bottom of a polystyrene well (Fig. 1A) . A mixed protein
lysate is added to the well and the target protein is “captured” onto the solid
phase by interaction with the capture antibody ( Fig. 1B ). A second antibody
recognizing a different antigenic determinant on the target protein is added
(Fig. 1C ). The resulting antibody-antigen-antibody sandwich is detected with
an enzyme-linked secondary antibody (Fig. 1D) followed by incubation with
a suitable enzyme substrate. Substrate hydrolysis results in a detectable color
change (Fig. 1E) and is proportional to the amount of captured protein. At
each step, the antibody sandwich is separated simply and effectively from
unbound conjugate by repeated washes. Sensitivity can be increased further by
secondary and tertiary immunogenic enhancement.
In this chapter, we describe standard protocols for protein detection in
lysates prepared from microdissected brain tissue using antibody-sandwich
ELISAs. To illustrate methods, cerebral changes in expression of connexin32
(Cx32) following chronic cocaine self-administration are presented. Connexins