Chemiluminescent Western Blot Detection using the FluorChem® HD2 Imaging System
Abstract
Digital image acquisition using the FluorChem HD2
is compared to film for chemiluminescent Western
blot detection. The results show that digital imaging
produces higher quality images at equal exposure
times and achieves greater dynamic range and detects
lower levels of protein than film. With the additional
advantages of reduced cost and time expended
per image, image archiving, and quantitative image
analysis, digital imaging is the method of choice for
chemiluminescent Western blot detection.
Introduction
Western blotting is a well characterized and widely used
method for quantification of protein abundance in cell
culture, tissue, serum and purified protein samples (1).
Chemiluminescence is the standard detection technique
exploiting the catalytic reaction of an enzyme and a
peroxide-based substrate to produce a light signal with
very low background as no illumination is required. The
enzyme is conjugated to a secondary detection antibody
that binds to the primary antibody specific to the protein of
interest. Chemiluminescence is widely used by researchers
because it is much less hazardous than radioactivity while
achieving equal performance.
Detection of chemiluminescence by exposure to film is
used commonly, but suffers from the well documented
limited linearity response and reciprocity failure of film.
The FluorChem HD2 and FC2 imaging systems apply CCD
cameras, fast lenses, and dark enclosures for quantitative
chemiluminescent detection and when used optimally are
powerful tools for Western blot analysis. Coupled with
Cell Biosciences ChemiGlow ® chemiluminescent substrate,
high quality images and accurate results are obtained with
fast imaging times. Furthermore the need for costly film
and film developers is eliminated.
Materials and Methods
Film vs FluorChem HD2 detection of chemiluminescent Western Blot
The performance of film and the HD2 were assessed
visually by imaging the same blot, first on the HD2 and
then by film.
A standard SDS-PAGE gel loaded with bacterial whole cell
lysates, purified transposase protein and standard protein
ladder was resolved and transferred to a blot using a
standard transferring technique. The blot was blocked,
incubated with rabbit anti-transposase primary antibody,
and then labeled with HRP conjugated goat anti-rabbit
antibody. The blot was treated with ECL (GE Healthcare)
and imaged using a FluorChem HD2 imaging system for
five minutes on normal resolution (no binning). The blot
was then exposed to film for five minutes and the film
developed.
Slot Blot Dilution Series
The capability of the FluorChem HD2 to quantify protein
amounts was assessed by imaging and analyzing a protein
dilution series.
A dilution series of an HRP conjugated antibody (goat antirabbit
IgG Horseradish Peroxidase Antibodies Inc, Davis,
CA) was prepared and deposited onto the surface of a low
autofluorescence blotting membrane (Millipore-FL) using a
filtration manifold apparatus (The Convertible, Gibco BRL,
Gaithersburg, MD). The concentration of protein in the
first band deposited was 1ng of total protein. Additional
dilutions were included as a 1:10 dilution series with a final
concentration of 100fg/band. The blot was treated with
ChemiGlow and imaged on the FluorChem HD2 imaging
system.
Results and Discussion
Image quality, linear dynamic range, and the capability to
detect low levels of protein are critical performance criteria
for chemiluminescent Western blot detection.
High image quality is required to visually assess the
separation of adjacent bands (resolution) and to see the
range of protein amounts (contrast). Film also required
an additional step of digitization of the original film using
white light illumination with a slight loss of contrast. The
FluorChem HD2 image of the chemiluminescent Western
blot, when judged by the clarity and contrast of the bands
as well as visualization of dim bands, was superior to that
of film.
Quantitative analysis of protein amounts requires a broad
linear measurable dynamic range and the capability to
detect low levels of protein. The FluorChem HD2 achieves
3.8 logs of linear measurable dynamic range (2) for longer
sustained (and useful) signal collection times compared to film based imaging (3). The signal from 500 fg deposited
protein was significantly above background variance. While
routine detection of low levels of proteins can also depend
on sample preparation and labeling conditions, these
results indicate that digital imaging is capable of detecting
low levels of protein.
When using the FluorChem HD2 small quantities of
protein can be detected simply by extending exposure
time while maintaining a broad linear response to signal
intensity. The detection of very faint bands by film can
require extended exposure times compared to the times
optimal for brighter signals due to reciprocity failure of film
with a loss of contrast and more rapid saturation of the
brighter signals.
In conclusion, detection of chemiluminescent Western
blots using the FluorChem HD2 imaging system produces
the high quality images and quantitative results required by
the most demanding Western blot experiments.
References
- Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular
Cloning. A laboratory manual. Cold Spring Harbor
Laboratory, N.Y.
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The FluorChem family of bioimaging
systems are designed for applications
in Chemiluminescent, Fluorescent, and
Colorimetric imaging. The FluorChem
line combines sensitivity, resolution and
dynamic range providing customers
the best in class
imaging capabilities for
Gel, Membrane and
Microplate based assays.
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