Multiplex Protein Detection

What is multiplexing in Western blots?

Multiplexing fluorescent Western blots, also referred to as multiplex fluorescence,  is the ability to detect multiple proteins simultaneously on the same blot. This allows you to get all the information you need at once. 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 (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).

Multiplex detection is possible by using two or more fluorescent dyes on an instrument that can excite and detect light from each dye
Multiplex detection is possible by using two ore more fluorescent dyes and an instrument that can excite and detect the light from each dye.

Advantages of multiplexing

With multiplexing, normalization and loading controls can be imaged at the same time and on the same blot as the sample. The ability to use different fluorophores enables visualization of proteins that are not well-separated electrophoretically. This allows 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. Unlike chemiluminescent Westerns, which are limited by the variable kinetics of the enzyme-substrate reaction, the amount of light emitted from fluorophores is highly consistent and directly proportional to the amount of protein on the membrane. This consistency means that fluorescent detection can provide a truly quantitative analysis of the proteins in question.

Multiplex fluorescent Western blot

How you can save time with multiplexing

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.


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!
Related Products
Azure Sapphire Biomolecular Imager

Laser-scanning system capable of detecting up to four proteins on the same Western blot

Azure 600 Western blot Imaging system

Flexible gel doc imaging system for multiplex Western blots

Looking for a full list of applications?


Related blog posts about multiplexing…

Antibody Labeling in the Lab

This blog post will cover antibody labeling in the lab. While imaging technology is advancing rapidly offering us scientists new and interesting ways to image

Read More
Shopping cart
There are no products in the cart!
Continue shopping