8 R&DMagazine February 2014 www.rdmag.com
The answer is “yes” if a couple of long-held myths are busted.
In an editorial cartoon that appeared in a recent issue of The Journal of Clinical Inves- tigation, a surgeon wields a scalpel over his patient. The caption reads: “Just a little nip
here and there. We don’t want it to look like it’s
had any work done.” The catch? The patient is
a western blot, and the doctor is presumably
making his patient look presentable for publication in a peer-reviewed journal.
The drawing’s appearance was “inspired”
by four articles that the journal pulled due
to alterations of western blot figures. Sometimes, these doctored figures make it through
the peer-review process, which can lead to
retraction of the published manuscript if
detected. In response to a rash of retractions
that seriously damaged the reputation of one
researcher and forced another to resign, publishers of the blog “Retraction Watch” wrote an
op-ed titled “Can We Trust Western Blots?”. In
the comments following the op-ed, one reader
wrote “I’d like to see western blotting follow
its northern cousin into oblivion.” What can
scientists do to restore trust in this once venerable—and now vulnerable—technique?
In an editorial article that accompanied the
cartoon, the journal editor reminded her read-
ers of “some experimental basics” for western
blotting. It’s a good idea to keep these things
in mind, as it’s not uncommon for researchers
to receive protocols from more senior labora-
tory members without critical examination. It
shouldn’t be difficult for scientists—who are,
after all, trained to ask the right questions—to
apply this skill to their own methods.
Know the limitations
Despite recent concerns about reliability of the
method and its results, western blotting is an
indispensable scientific tool used to quantify
relative protein levels, with applications spanning
from clinical diagnostics to answering fundamental questions in life science. However, as with any
powerful analytic method or technique, western
blotting has inherent limitations.
Journal editors and scientific peers are now
acknowledging this, while also recommending
guidelines and testing controls to promote
clearer and more reproducible results. It’s clear
that researchers must have a firm understanding of these guidelines, both in theory and in
practice, to ensure the integrity of their results.
With such a time- and resource-intensive
technique, awareness of the western blotting
process and its limitations are critical.
Two important steps for producing reliable
western blot data are normalization and signal
detection. Long-held misperceptions—call
them “myths”—surround these steps and hinder researchers’ ability to use western blots as a
meaningful tool for the quantification of relative protein expression levels. Addressing these
so-called myths will go a long way to restoring
trust in western blots.
Myth #1: Using housekeeping proteins
is the best normalization method
When comparing relative protein expression
levels in western blots, normalization methods
are commonly used to control for technical
errors and inconsistencies that arise during
sample preparation, loading and transfer steps.
For instance, inherent variations in transfer
efficiencies may result in two- to four-fold
increases or decreases in the signal between
gel lanes. To correct for user inconsistencies
that impact signal intensity, scientists employ
normalization techniques. The most popular is
the use of housekeeping proteins (HKPs) such
as GADPH, beta-actin and tubulin. However,
it is crucial that researchers have a good understanding of the normalization technique and
assay parameters to ensure validity.
Drawbacks of housekeeping proteins
HKPs are thought to accumulate at constant
levels under all conditions and in all cell types,
because they’re constitutively expressed and
maintain cell viability. This may not be true,
though. Studies indicate that housekeeping protein expression can vary according to conditions
such as tissue type, disease state, sample preparation and the environment. HKP characterization
is often bypassed because validating the use and
stability of HKPs per application adds significant cost and time to each experiment.
A second, often overlooked, drawback to using
HKPs is signal saturation. Target proteins are
generally only present in low quantities. Thus,
scientists must overload the sample to quantify
low-level proteins. This technique may make proteins more visible, but it can also spell trouble.
Graduate students in Dr. Ji-Joon Song’s laboratory in the Korea Advanced Institute of Science and
Technology use Bio-Rad’s ChemiDoc MP imaging system to image stain-free gels and blots and
quantitate chemiluminescent western blots. Images: Bio-Rad Laboratories