Multiplex Protein Detection

What is multiplexing in Western blots?

Multiplexing fluorescent Western blots (Figure 1), also referred to as multiplex fluorescence, refers to the “multiplex” ability to detect multiple proteins simultaneously on the same Western blot. This allows researchers to get all the information they need at once.

Multiplex fluorescent Western blot from Azure Biosystems imager
Figure 1. Digital images of 4-color multiplex Western Blot. Using distinct fluorescent and near-infrared targeting antibodies can detect each wavelength and merge them into a four-color multiplex image. No background noise or bleeding between channels. Image captured with the Sapphire Biomolecular Imager

Fluorescent Western blotting uses secondary antibodies directly conjugated to fluorescent dyes. Fluorophores can be chosen based on their specific excitation and emission spectra, enabling multiplexing (Figure 2) 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 2. 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.

Table 1. Advantages and disadvantages of fluorescent multiplex protein detection

AdvantagesDisadvantages
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 laterNeed an imager with multiplex protein detection
Fluorescent detection can provide a truly quantitative analysis of proteins in question
Saves time

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
Figure 3. Multiplex fluorescent imaged with an Azure Imager using Cy3 and Cy5 channels.

How multiplexing Western blots saves time

Multiplexing your Western blots can save you time in a multitude of ways: avoiding saturation problems by using sophisticated detection technology and letting you have the option of arching your Western blots by using fluorescent dyes.

The newer generation of imaging systems, like the Sapphire FL, 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.

Fluorescent Westerns are typically visualized using a digital imager rather than X-ray film (Figure 1). Figure 3 shows a fluorescent multiplex Western blot imaged using an Azure 600 imager. Finally, fluorescent dyes are relatively stable. Western blots can be archived and imaged months after the initial experiment as long as precautions are taken to avoid photo-bleaching of the fluorophores.

Imaging systems capable of multiplex Western blots

The Sapphire FL is capable of detecting up to four proteins on the same Western blot, which allows you to save money on each experiment. It uses 4 fluorescence channels to easily quantify overlapping bands.

Sapphire FL biomolecular imager
The Sapphire FL Biomolecular Imager scans with up to three optical modules at once. Quickly and easily swap one optical module for another to create a four-channel fluorescent image. Detect up to four proteins on the same Western blot.

Multiple channels simplify normalization, which eliminates the need for stripping and re-probing. It offers simultaneous multichannel scanning to reduce your total imaging time. Learn more about the multiplex capabilities of the Sapphire FL here.

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