The FluorChem® Q and ECL Plex™: Sensitive and Quantitative Imaging of Multicolor Fluorescent Westerns
Multiplex fluorescent detection allows the detection and analysis
of multiple proteins on a single Western blot. This approach
facilitates quantification of proteins via the use of an internal
reference protein, removing the need to strip and reprobe a blot. The
FluorChem® Q provides a sensitive means to acquire high-quality
images of multicolor fluorescent Westerns. This report demonstrates
the sensitivity and linear range which can be achieved in using
the FluorChem® Q with ECL Plex™ labeled Westerns, and the
compatibility of the FluorChem® Q with multicolor Western blots.
Multiplex analysis, or multiple fluorescent channel imaging, allows for
the simultaneous analysis of multiple RNA samples on a microarray,
or multiple antigen locations in a cell via fluorescence microscopy.
Recently, this technology has become available for Western blotting, a
powerful tool for the protein chemist.
The most commonly used detection method for Western blotting,
chemiluminescence, is a well-characterized technique for sensitive
detection of proteins (1). However, this method can present limitations
in applications where multiple proteins are of interest. If two proteins
of interest are similar in size and migrate closely together on a gel, as is
the case for phosphorylated isoforms, only one protein can be assayed
at a time. The blot must be stripped and reprobed independently for
each protein, which is time-consuming and may result in protein loss.
Alternatively, duplicate blots may be run, which consumes precious
When quantifying a protein across multiple samples, a housekeeping
or reference protein is used to control for differences in gel loading.
Frequently this also requires stripping and reprobing of the
chemiluminescent Western blot, unless the reference protein is well
differentiated in size from the protein of interest. If the housekeeping
protein and the protein of interest are probed simultaneously on a
chemiluminescent blot, and the proteins have very different abundances,
a large difference in signal strength between the two proteins may
result. Under such conditions, multiple exposures of the Western blot
to film may be required to identify conditions under which one signal is
not saturated. Direct fluorescent labeling of the secondary antibodies,
combined with fluorescent light detection, overcomes these limitations.
Multiple proteins can be assayed on one blot, regardless as to whether
they overlap or are in different relative abundance.
The ECL Plex™ Western Blotting Detection System (GE Healthcare)
uses CyDye-conjugated secondary antibodies. The direct fluorescent
detection of the antibodies allows for excellent sensitivity, linearity, and
dynamic range (2). Additionally, direct fluorescent detection allows
for multiplex applications in which multiple proteins can be detected
simultaneously on the same blot by the use of secondary antibodies
labeled with different fluorescent dyes (Figure 1). Each dye is chosen
to have unique spectral properties so that, with proper selection of
excitation and emission wavelengths, they can be independently excited
The FluorChem® Q imaging system is designed to acquire high quality
images from fluorescently labeled, multicolor westerns. Three excitation
and three emission filters on the FluorChem® Q provide three unique
spectral detection channels, optimized for the most commonly used
fluorescent reagents. The compatibility of the FluorChem® Q filters
with the excitation and emission spectra of the Cy2, Cy3 and Cy5 dyes
used in the ECL Plex™ system is shown in Table I.
Important to the collection of fluorescence images useful for quantitative
analysis is a large dynamic range, accomplished through a combination
of high sensitivity and low background noise. The FluorChem® Q has
a high power illumination field with consistent intensity across its large
field of view, allowing for uniform imaging across the entire blot. An
integrated 4.2 megapixel, peltier-cooled camera collects high-resolution
images while minimizing noise, and a F 0.95 fixed lens provides fast
image acquisition, shortening analysis time and helping prevent
In this article, we demonstrate the performance of the FluorChem® Q
in detecting and quantifying multicolor Western blots labeled with the
ECL Plex™ fluorescent detection system. The linearity, dynamic range,
and detection limit of Transferrin detected with a Cy5 labeled antibody
are presented, and the ability to image three proteins on a single blot
using three different fluorescent channels is demonstrated.
Electrophoresis and transfer. Proteins were separated on 10-20%
polyacrylamide Tris-HCl gels (Bio-Rad 161-1124), using a standard
Tris-Glycine-SDS buffer system in the Mini-PROTEAN Tetra
Cell from Bio-Rad. Transfers were conducted in Tris-Glycine buffer
containing 12% methanol.
Experiment 1: Two-fold serial dilutions of Transferrin were prepared,
spanning the range from 0.6 pg to 9.3 ng, and loaded on the gel in a final volume of 10 µl. Electrophoresis was carried out at 90 V for two
hours. After separation, proteins were transferred to PVDF membrane
(Immobilon-FL, Millipore IPVH00010) overnight at 15V.
The membrane was washed with PBS containing 0.05% Tween-20
(PBST) for 5 minutes. The membrane was then blocked for one hour at
room temperature in a 1:10 dilution of BSA blocker (Pierce 18587) as
per manufacturers instructions.
The blot was incubated with the primary antibody, Rabbit-anti-
Transferrin (Abcam ab1223-1) at 5 µg/ml for two hours at room
The blot was washed five times with Pierce wash buffer (1860498)
diluted 1:30 as per manufacturers instructions.
The blot was incubated one hour at room temperature with the
secondary antibody, Cy5-labeled Goat-anti-Rabbit IgG (GE Healthcare
PA45012V) diluted 1:2500 in wash buffer.
The blot was then washed six times in Pierce wash buffer for five minutes
each. After a final rinse in PBS, the blot was imaged while still damp,
approximately one hour after the PBS rinse. The blot was placed on a
low-fluorescent plastic sheet on the sample holder for imaging.
Experiment 2: All samples were loaded in equal volumes (10 µl). Each
lane contained 30 ng of Human IgG, corresponding to approximately
20 ng of heavy chain. Two-fold serial dilutions of Transferrin, and of
α1-fetoprotein (AFP), were loaded on the gel as follows:
Separation was carried out at 150 V for one hour,
After separation, proteins were transferred to a PVDF membrane
(Immobilon-FL, Millipore IPVH00010). Transfer was conducted at
100 V for one hour with an ice pack inserted in the tank.
The membrane was washed with PBS containing 0.05% Tween-20
(PBST) for 5 minutes. The membrane was then blocked for one hour
at room temperature in a 2% solution of ECL Advance blocking agent
(GE Healthcare CPK1075) dissolved in PBST.
After blocking, the membrane was washed with PBST for 5 minutes
and incubated with a mixture of primary antibodies, containing Rabbitanti-
Transferrin (Abcam ab1223-1) and Mouse-anti-AFP (Meridian
Life Sciences H45301M) antibodies, each diluted at 1:5000 in PBST.
The membrane was then subjected to two brief, one 15 minute, and four
five-minute washes in PBST.
The membrane was then incubated with a mixture of fluorescently
labeled secondary antibodies for one hour at room temperature. The
secondary antibodies used were Cy3-Goat-anti-Mouse IgG at 1:2500
dilution, and Cy5-Goat-anti-Rabbit IgG at 1:2500 dilution (both
from GE Healthcare). A Cy2-Goat-anti-Human IgG antibody was
also added at 1:2500 dilution, to detect Human IgG directly. The
Cy2-Goat-anti-Human IgG antibody was prepared from non-labeled
antibody (Vector Labs AI-3000) using the Cy2 labeling kit from GE
(GE Healthcare PA32000).
After hybridization, the membrane was washed three times with PBST
for five minutes each, once with PBS for five minutes, then briefly rinsed
with water and dried at room temperature.
The dry membrane was imaged with the FluorChem® Q imaging
system at 1.7x magnification. The membrane was positioned on the
Single channel images of each of the three fluorescent dyes were acquired
on the FluorChem® Q. The spectral characteristics of the filter sets used
are shown in Table 1. The exposure times were adjusted manually for
each of the three channels.
Cell Biosciences products used
- FluorChem® Q
Other materials required
- 10-20% linear gradient Ready Gel (Bio-Rad 161-1124)
- Immobilon-FL PVDF membrane (Millipore IPVH00010)
- ECL Advance blocking agent (GE Healthcare CPK1075)
- BSA blocker (Pierce 1858753)
- Pierce wash buffer (Pierce 1860498)
- Rabbit-anti-Transferrin antibody (Abcam ab1223-1)
- Mouse-anti-AFP (Meridian Life Sciences H45301M)
- ECL Plex™ goat-α-mouse IgG-Cy3 (GE Healthcare PA43009)
- ECL Plex™ goat-α-rabbit IgG-Cy5 (GE Healthcare PA45011)
- Goat-anti-Human IgG (Vector Labs AI-3000)
- Cy2 Ab Labeling Kit (GE Healthcare PA32000)
- ECL Advance blocking agent (GE Healthcare CPK1075)
- Mini PROTEAN Tetra cell (Bio-Rad 165-8004)
- Tris-Glycine running buffer
- Tris-Glycine transfer buffer
- PBS buffer
- PBST buffer
Results and Discussion
Experiment 1: The performance of the FluorChem® Q was tested by
preparing a Western blot of a dilution series of Transferrin, a protein
normally present in Human serum. An image of the Western blot,
acquired on the FluorChem® Q with a 16 second exposure is shown
in Figure 2. A measurable linear dynamic range of over 2000-fold was
achieved (Figure 2). Exposing a duplicate blot for 256 seconds gave a
lower limit of detection of 0.6 pg (Figure 3)
Experiment 2: The compatibility of the FluorChem® Q and ECL Plex
fluorescent western blotting system when imaging multicolor Western
blots was tested by preparing a Western blot containing serial dilutions
of two proteins, Transferrin and AFP, as well as a constant level of
a third protein, Human IgG. The three proteins were imaged in one
imaging protocol, each in a different fluorescent channel. Though each
of the proteins can be imaged independently, the images captured in
each of the three fluorescent channels can be merged to allow the three
proteins to be visualized simultaneously. Such a merged image depicting
each of the three fluorescent channels is shown in Figure 4a. The power
of multiplexing is demonstrated by this image, in which Transferrin
(73-76 kDa) and AFP (70kDa) are easily distinguished.
The single channel images captured in the red channel (Transferrin,
Figure 4b), and green channel (AFP, Figure 4c), demonstrate the large
dynamic range and low limit of detection achieved for each protein and
dye combination in a true Western blot experiment. The blue channel,
IgG, can be used in later analysis as a loading control (Figure 4d).
The linear range and detection limit obtained for a given Western blot
experiment will depend on multiple experimental factors including the
nature of the protein of interest, the efficiency of transfer of the protein
from gel to membrane, the selection of the primary and secondary
antibodies, and optimization of the antibody hybridization conditions.
The results presented in this report are specific to the materials and
conditions described herein.
The FluorChem® Q provides a powerful means to image and quantify
multicolor fluorescent Western blots labeled using the ECL Plex™
system. The sensitivity of the system is reflected in a detection limit
of 0.6 pg of Transferrin. With an optimized exposure time, the
fluorescence intensity of Transferrin staining was linear over three
orders of magnitude, covering a range of protein amounts from 4.5 pg
to 9.6 ng, which encompasses the range commonly seen in Western
The FluorChem® Q also performed in multiplex analysis, acquiring
images of three proteins, detected with three unique fluorophores on
a single blot.
The linearity of the response over a large dynamic range, combined with
the ability to detect multiple proteins simultaneously on the same blot,
demonstrates the potential for accurate protein quantification, with a
minimization of time and sample usage.
- Patton, Wayne F. (2000) “A thousand points of light:
The application of fluorescence detection technologies to
two-dimensional gel electrophoresis and proteomics”
Electrophoresis. 21: 1123-1144.
- GE Healthcare Application Note 28-4015-40 AB “Multiplex
protein detection using the ECL Plex™ fluorescent Western
Cell Biosciences gratefully acknowledges the researchers at Advansta
Corporation in Menlo Park, California, for the information contained
in this Tech Note.
The FluorChem® Q Imaging System provides the sensitivity of chemiluminescence and the quantitative power
of fluorescence in one easy-to-use instrument. Designed with fast lens technology, the peltier cooled camera
captures high-resolution images with a linear dynamic range that outperforms film, and with speeds 10X faster
than a laser scanner. Equipped with three integrated excitation sources for multicolor westerns, the FluorChem®
Q is compatible with Cy dyes, Alexa dyes, Qdots, and chemiluminescent western blotting kits. Additionally,
the FluorChem Q provides the flexibility to image fluorescently stained DNA gels, as well as Coomassie and
silver stained protein gels.
For more information, visit us at: http://www.cellbiosciences.com