<p class="first" id="P1">Western blot is routinely used to quantify differences in
the levels of target proteins
in tissues. Standard methods typically use measurements of housekeeping proteins to
control for variations in loading and protein transfer. This is problematic, however,
when housekeeping proteins also are affected by experimental conditions such as injury,
disease, and/or gonadal hormone manipulations. Our goal was to evaluate an alternative
and perhaps superior method for conducting Western blot analysis of brain tissue homogenates
from rats with distinct physiologically relevant gonadal hormone states. Tissues were
collected from the hippocampus, frontal cortex, and striatum of young adult female
rats that either were ovariectomized to model surgical menopause, or were treated
with the ovatotoxin 4-vinylcyclohexene diepoxide (VCD) to model transitional menopause.
Tissues also were collected from rats with a normal estrous cycle killed at proestrus
when estradiol levels are high, and at diestrus when estradiol levels are low. Western
blot detection of α-tubulin, β-actin, and GAPDH was performed and were compared for
sensitivity and reliability with a fluorescent total protein stain (REVERT®). Results
show that the total protein stain was much less variable across samples and had a
greater linear range than α-tubulin, β-actin, or GAPDH. The stain was stable and easy
to use, and did not interfere with the immunodetection or multiplexed detection of
the housekeeping proteins. In addition, we show that normalization of our data to
total protein, but not to GAPDH, revealed significant differences in α-tubulin expression
in the hippocampus as a function of treatment, and that gel-to-gel consistency in
measuring differences between paired samples run on multiple gels was significantly
better when data were normalized to total protein than when normalized to GAPDH. These
results demonstrate that the REVERT® total protein stain can be used in Western blot
analysis of brain tissue homogenates to control for variations in loading and protein
transfer, and provides significant advantages over the use of housekeeping proteins
for quantifying changes in the levels of multiple target proteins.
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