Multiplex Fluorescent Westerns

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.

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.

Multiplex capabilityCan be less sensitive than chemiluminescence
Increased quantitative accuracyMembranes' auto fluorescence can increase background
Fuorescent label stability allows blots to be
stored and re-imaged later

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

The best imager for multiplex fluorescent Westerns

Fluorescent Westerns are typically visualized using a digital imager, rather than X-ray film (Figure 2). The newer generation of imaging systems often contain sophisticated cameras that exhibit a broader dynamic range than film, thus avoiding the signal saturation problems that limit the dynamic range of film. Finally, 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.


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Featured Publication

Check out this multiplex fluorescent Western paper from the NIH that researches implications for SARS-CoV-2 infection after repeated ethanol exposure.

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