Multiplex Fluorescent Western blots

What is fluorescent Western blotting?

Fluorescent Western blotting uses secondary antibodies directly conjugated to fluorescent dyes. Fluorophores for fluorescent Western blotting can be chosen based on their specific excitation and emission spectra, enabling multiplexing (detection of multiple proteins simultaneously, Figure 1) for faster, more efficient studies. One of the biggest advantages of fluorescent detection versus chemiluminescence is the ability to use more than one antibody per assay (see the Table).

Diagram of how multiplex fluorescent Westerns can detect two proteins in two spectrally different channels
Figure 1. Multiplex detection is possible by using two or more fluorescent dyes and an instrument that can excite and detect the light from each dye.

With multiplexing, normalization and loading controls can be imaged at the same time and on the same blot as the sample. In addition, the ability to use different fluorophores enables visualization of proteins that are not well-separated electrophoretically, for more convenient imaging of the same protein with and without post-translational modifications.

Why would you need to visualize multiple proteins at once (Table 1). Convenience is an obvious benefit to multiplexing, as loading controls are often used for Western blot data. By using different fluorescently labeled secondary antibodies for the protein of interest along with the loading control, clear and quantifiable results are ready to be analyzed immediately, even if the proteins are close in molecular weight. Quantitation is also increasingly desirable when publishing protein expression data. What if you have two different proteins of interest, such as phosphorylated and non-phosphorylated version of a protein? In order to include a loading control for accurate comparison of different lanes, a third color would be necessary. Conveniently, multiplex Western blotting is possible and easy with AzureSpectra labeled antibodies and the Azure 600 Imaging System.

Multiplex capability: multiple samples can be analyzed under the same experimental conditions.Can be less sensitive than chemiluminescence
Increased quantitative accuracyMembranes' auto fluorescence can increase background
Fluorescent label stability allows blots to be stored and re-imaged later
More readout consistency using fluorescent dyes vs. the variability of an enzymatic reaction
Cost and time savings

Table 1. Advantages and disadvantages of multiplex fluorescent Western blots

Fluorescent Western blots are the gold standard for quantitative Westerns. Chemiluminescent Westerns are limited by the variable kinetics of the enzyme-substrate reaction; however, the amount of light emitted from fluorophores is highly consistent. The amount of light is directly proportional to the amount of protein on the membrane. This consistency means fluorescent detection can provide a truly quantitative analysis of the proteins in question.

Two color multiplex fluorescent western blot
Multiplex fluorescent Western blot imaged with an Azure Imager using Cy3 and Cy5.

Using AzureSpectra Fluorescent Secondary Antibodies

AzureSpectra Fluorescent Secondary antibodies are labeled with fluorophores that emit light in visible and near-infrared wavelengths. The AzureSpectra 490, 550, 650, 700 and 800 secondary antibodies offer unparalleled sensitivity and performance for immunoblotting applications when used in conjunction with Azure Western blotting imaging systems.

maximum (nm)
Maximum (nm)

The Azure 600 Imager and AzureSpectra antibodies allow for quick and easy detection of three different proteins in the same samples on the same Western blot. Accurate quantitative analysis of your fluorescent western blot can be performed immediately after imaging.

Figure 3 shows the digital image of the Western blot labeled with three different primary antibodies (anti-tubulin, anti-beta actin, and anti-GAPDH) and fluorescently labeled secondary antibodies. The individual channels are shown in grayscale and all three are merged in the three-color image (upper left). The ability to visualize three proteins at once increases the potential information that can be obtained from individual Western blot experiments. With more information, better quantitative analysis can beperformed – strengthening your data and your science.

Digital image of 3-color fluorescent multiplexed Western blot
Figure 3. Digital image of 3-color fluorescent multiplexed Western blot using Azure Biosytems c600 imager. Lanes (from left to right) loaded with 1, 2, 5, 10, 20 µg HeLa cell lysate. Probed for tubulin (top), beta actin (middle) and GAPDH (bottom). The following settings were used: Light sources 6/7/4; Exposure time 1s/13s 204ms/677ms; Filter positions 6/7/4; Aperture 6400; Focus 5000/5250/5000; bin level 1x1.
Excitation /
Azure Imaging
Rat anti-TubulinAzureSpectra Goat-anti-rat 800785 nm / 815 nmIR800
Rabbit antibeta-actinAzureSpectra Goat-anti-rabbit 700660 nm / 720 nmIR700
Mouse antiGAPDHAzureSpectra Goat-antimouse 550551 nm / 565 nmCy3

Instructions for use, tips, troubleshooting and further information are available in the AzureSpectra AzureSpectra NIR Secondary Antibodies Product Insert instruction manual and Transition from Chemiluminescent to Fluorescent Western Blots instruction manual.

AzureSpectra 490 Fluorescent Secondary Antibodies

Catalog NumberDescriptionSize
AC2206Goat-anti-rabbit 490250uL at 1mg/mL
AC2207Goat-anti-mouse 490250uL at 1mg/mL
AC2208Goat-anti-human 490250uL at 1mg/mL
AC2209Goat-anti-chicken 490250uL at 1mg/mL
AC2210Goat-anti-rat 490250uL at 1mg/mL
AC2211Goat-anti-guinea pig 490250uL at 1mg/mL
AC2217Donkey-anti-goat 490250uL at 1mg/mL

AzureSpectra 550 Fluorescent Secondary Antibodies

Catalog NumberDescriptionSize
AC2158Goat-anti-rabbit 550250uL at 1mg/mL
AC2159Goat-anti-mouse 550250uL at 1mg/mL
AC2160Goat-anti-human 550250uL at 1mg/mL
AC2161Goat-anti-chicken 550250uL at 1mg/mL
AC2162Goat-anti-rat 550250uL at 1mg/mL
AC2163Goat-anti-guinea pig 550250uL at 1mg/mL
AC2164Donkey-anti-goat 550250uL at 1mg/mL

AzureSpectra 650 Fluorescent Secondary Antibodies

Catalog NumberDescriptionSize
AC2165Goat-anti-rabbit 650250uL at 1mg/mL
AC2166Goat-anti-mouse 650250uL at 1mg/mL
AC2167Goat-anti-human 650250uL at 1mg/mL
AC2168Goat-anti-chicken 650250uL at 1mg/mL
AC2169Goat-anti-rat 650250uL at 1mg/mL
AC2170Goat-anti-guinea pig 650250uL at 1mg/mL
AC2171Donkey-anti-goat 650250uL at 1mg/mL

Due to low background autofluorescence in the near-infrared region, AzureSpectra 700 and 800 secondary antibodies can produce higher signal-to-noise ratios.

AzureSpectra 700 Fluorescent Secondary Antibodies

Catalog NumberDescriptionSize
AC2128Goat-anti-rabbit 700250uL at 1mg/mL
AC2129Goat-anti-mouse 700250uL at 1mg/mL
AC2130Goat-anti-human 700250uL at 1mg/mL
AC2131Goat-anti-chicken 700250uL at 1mg/mL
AC2132Goat-anti-rat 700250uL at 1mg/mL
AC2133Goat-anti-guinea pig 700250uL at 1mg/mL
AC2156Donkey-anti-goat 700250uL at 1mg/mL

AzureSpectra 800 Fluorescent Secondary Antibodies

Catalog NumberDescriptionSize
AC2134Goat-anti-rabbit 800250uL at 1mg/mL
AC2135Goat-anti-mouse 800250uL at 1mg/mL
AC2136Goat-anti-human 800250uL at 1mg/mL
AC2137Goat-anti-chicken 800250uL at 1mg/mL
AC2138Goat-anti-rat 800250uL at 1mg/mL
AC2139Goat-anti-guinea pig 800250uL at 1mg/mL
AC2157Donkey-anti-goat 800250uL at 1mg/mL

The best imager for multiplex fluorescent Westerns

In fluorescent Western blotting, the secondary antibody is directly conjugated to a dye, which is excited by light. The emitted light is detected by a digital imager such as an Azure 400, rather than X-ray film (Figure 2) and digitized for data analysis. Multiple proteins can be detected simultaneously by using secondary antibodies conjugated to different dyes with non-overlapping spectral emissions.

The newer generation of imaging systems often contain sophisticated CCD cameras that exhibit a broader dynamic range than film, thus avoiding the signal saturation problems that limit the dynamic range of film. In addition, fluorescent dyes are relatively stable; blots can be archived and imaged months after the initial experiment as long as precautions are taken to avoid photo-bleaching of the fluorophores.

Two scientists looking at multiplex fluorescent Western blot on Azure 600 Western blot imager
Revolutionizing the way you Western blot! Azure Imagers are high performance Western blot imaging systems capable of NIR fluorescence, visible fluorescence, and chemiluminescence.

Azure imaging systems offer:

  • Multiplex fluorescence capabilities

  • Speed and sensitivity of X-ray film

  • High-resolution imaging perfect for publications
  • IQOQ Validation

Learn more about the Azure Imaging Systems, including full product specifications, user manuals and resources.

New to fluorescent Western blotting?

Fluorescent Western blots can be tricky. Don’t worry, we’ve got you covered. Azure’s Fluorescent Demo Kits contain everything you need to perform a single-color fluorescent Western using your own primary antibody and protein sample of your choice. Each kit contains enough material for 1 Western blot.


New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!
Products for Western blotting
NEW! Azure Sapphire FL Biomolecular Imager

Able to detect up to 4 multiplexed fluorescent reactions per Western blot

Designed for multicolor
fluorescent Western blotting, with two NIR and 3 visible fluorescent channels

Three-color fluorescent detection for dyes in the visible range

Featured Publication

Check out paper from the NIH that uses multiplex fluorescent Western blots to research implications for SARS-CoV-2 infection after repeated ethanol exposure.

Additional Resources for Multiplex Fluorescent Westerns

Looking for a full list of applications?

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