When saturated, the HKP levels are no
longer within the linear dynamic range for
immunodetection, preventing accurate quantification. To alleviate this overloading concern,
researchers should first characterize and then
select the HKP in which the protein signal is
linear and within the system’s dynamic range.
This involves individually optimizing each
HKP with respect to antibody dilutions, incubation times and image settings.
Even if HKPs are properly validated for use
under the relevant experimental conditions
and optimized so they are present at levels
within the linear dynamic range of quantitation, the process of using them—either by
stripping and re-probing or via multiplex fluorescent blot detection—can be daunting. The
strip and re-probe method is time consuming,
and the stripping process inevitably removes
some level of antigen, thereby compromising
downstream results. Although a more elegant solution, multiplex fluorescent western
blotting requires optimization of blocking
reagents, antibody concentrations and incubation times, while users need to be mindful of
challenges like antibody cross-reactivity.
A better alternative:
Total protein normalization (TPN)
Use of total protein normalization (TPN) as a
loading control, another normalization meth-
od, is less affected by the limitations described
in HKP normalization. Studies on HKPs have
shown that total protein staining of the gels or
blots is better suited than HKPs to correct for
differences in loading. Also, TPN’s wider linear
dynamic range exhibits better linearity at the
lower, more relevant protein levels ( 10 to 50 µg)
used in western blotting. The TPN technique
is so well matched to this need that the target
protein is directly normalized against the total
protein concentration in each lane, making this
technique more universal and accurate than
HKP normalized experiments.
Aldrin Gomes, an asst. prof. at the Univ. of
California-Davis and a leading advocate for
improving the reliability of western blots, hopes
that a decade from now most researchers will be
using TPN as their normalization method.
The major limitation to conventional TPN
is that the blots are stained with total protein
stains, such as Sypro Ruby or Ponceau S.
Although the staining process provides better
sensitivity, it adds complexity, cost and time,
and introduces time-dependent variables.
A recent development in TPN, known as the
“stain-free” approach, possesses even greater
sensitivity at low protein levels while reducing
the complexity, cost and time associated with
staining. The technology uses a tri-halo compound that binds to proteins during gel electrophoresis, and allows researchers to directly
visualize and quantify proteins both in gels
and on blots within minutes. As this innovative
approach eliminates the staining step, stain-free TPN is more cost effective, time effective
and robust than its conventional counterpart.
Myth #2: X-ray film is the best method
for detecting western blots
X-ray film remains the most widely accepted
and commonly used detection technique for
visualizing chemiluminescent blots, which are
the most popular type of western blot. This is
due to x-ray’s high sensitivity, and resolution
Yet one major challenge of x-ray film is
saturation; it’s easily saturated by chemiluminescent signals from the blot, which precludes
quantitation. In fact, it’s this ease of signal saturation that gives scientists the (false) idea that
film is more sensitive than digital imaging.
Also, it’s not obvious when oversaturation
has occurred. When working with weakly
expressed proteins, researchers often go with
longer exposure times at the expense of oversaturating the stronger expression signals, such
as those that result from an overabundance of
HKPs. To prevent saturation effects, researchers must characterize x-ray film to identify its
linear dynamic range for detection of a particular antibody under the relevant testing conditions to prevent saturation effects.
Recently developed digital imaging methods
provide a wider linear dynamic range and
lower limits of detection, thereby addressing
the saturation issue. A recent study found that
using a two-fold dilution series of a protein
lysate, along with a digital imaging system,
resulted in a dynamic range nearly an order of
magnitude greater than that with film (0.04 to
2. 5 ng versus 0.04 to 0.31 ng) for the protein
which they probed. It’s also worth noting that
the costs of advancements in digital imaging
increasingly make this detection method more
reliable and affordable. The costs are now very
comparable to film, especially when factoring
in the price of the developer, ongoing running
costs and the increasing price of film.
Advances in western blotting over the past
decade, in both normalization and detection
methods, mean researchers can quantify west-
ern blots more reliably. It’s up to the scientist
to critically re-examine the tools they currently
use and make choices that will improve their
results. Once this happens, confidence in west-
ern blot data will inevitably follow.
— Ning Liu
Senior Product Manager,
Laboratory Separation Div.
Bio-Rad Laboratories Inc.
Digital imaging systems are able to capture
the less-intense signals that are missed by
film without compromising stronger signals to
saturation. Pictured: Shannamar Dewey, a Univ.
of California-Davis graduate student in the
Gomes laboratory, uses Bio-Rad’s ChemiDoc
MP system to image her western blot.
Stain-free technology enables researchers to
reap the benefits of total protein staining to
normalize their chemiluminescent western
blots without any of its drawbacks. Pictured:
Screenshot of a multi-channel image of stain-
free (left) and chemiluminescent blot images
taken from Bio-Rad’s Image Lab image analy-