Analysis of Protein Expression in Brain Tissue by ELISA - Drugs of Abuse: Neurological Reviews and Protocols - page 287

change can be replaced with precise quantitation of the protein concentration
in each sample ( see Note 2 ).

4. Notes

1. Alternative protocols for protein extraction include the use of Trizol Reagent
(Invitrogen, Burlington, ON) permitting simultaneous extraction of RNA and
protein from the same tissue sample and minimizing the number of animals
required to perform both RNA and protein studies (4) . However, because Trizol-
extracted protein is solubilized in 1% SDS, care should be taken to ensure that
the fi nal concentration of SDS does not exceed 0.5% when samples are diluted
in coating buffer ( Subheading 3.3.2., step 1 ). Stripping of capture antibody
from the microtiter plates and loss of antibody-antigen complex can occur at
elevated SDS concentrations.

2. The capture antibody can be either monoclonal or polyclonal and is diluted to
a concentration between 0.5 and 10 µ g/mL. As a rule, we have observed that

2.5 g/mL is a convenient starting concentration and that sensitivity does not

signifi cantly increase when concentrations exceed 10

µ

g/mL. If the capture and
detection antibodies have not previously been used for ELISA analysis, it may
be necessary to establish the limits of sensitivity using serial dilutions of both
reagents detecting a known positive control. Note that the sensitivity of the

µ

Fig. 2. (previous page) Analysis and interpretation of antibody-sandwich ELISA
results. (A) Distribution of Cx32 protein in the dorsal striatum, nucleus accumbens,
and globus pallidus/ventral lateral striatum (minus nucleus accumbens) in drug-naive
animals. Data represent the mean absorbance ± SEM of two independent experiments.
Note that the expression levels of Cx32 vary under control conditions. The limit of
sensitivity (i.e., the average of wells labeled 0 g of protein in Table 2 ) is illustrated by

the line at the bottom of the graph. Absorbances below this limit can not be distinguished
from nonspecifi c binding ( see Table 1). (B) Alterations in Cx32 expression following
withdrawal from chronic cocaine self-administration. Data are expressed as the mean
absorbance ± SEM of two independent experiments. The limit of sensitivity is indicated
by the horizontal line at bottom of the graph. As presented, it is diffi cult to compared
changes in Cx32 expression following cocaine withdrawal. (C) -Fold changes in Cx32
expression in the dorsal striatum, nucleus accumbens, and globus pallidus/ventral lateral
striatum during cocaine withdrawal. Standardization permits a clearer comparison of
trends in Cx32 expression at various time points after cocaine self-administration. Note
that relative protein expression does not change in the dorsal striatum during cocaine
withdrawal but that expression decreases over time to below that observed in drug-
naive animals in the nucleus accumbens. These data replicate previously published
results (4) . In the globus pallidus/ventral lateral striatum, a sharp increase in Cx32
expression is observed immediately after cocaine withdrawal followed by a gradual
decrease to basal protein levels.

µ