Multiplex Approach to Fluorescent Western Blotting

This article discusses the benefits of using fluorescent western blotting (WB) to detect multiple targets using the same blot simultaneously.

The two greatest advantages of using multiplexing processes during fluorescent western blotting are:

The ability to quantitatively assess the relative abundance of a protein

Western blotting allows researchers to compare the relative concentration of one protein against another in the same preparation, for example, to compare the phosphorylated form of the protein with the total protein present in the solution.

The ability to complete normalization without having to change the gel

Multiplexing in fluorescent WB allows the band intensity to be normalized using an internal control in the same blot, without having to strip it and then reprobe it.

Fluorescent WB Allows Quantification and Multiplexing

The quantitative nature of fluorescent tests compared to those which are based on the use of enzymes, like HRP, allows for more precise and simplified normalization, using an internal control such as a housekeeping gene.

In addition, fluorescent WB has the largest dynamic range when compared to other fluorescent tests, facilitating the detection of proteins at both high and low abundance levels in the same experiment.

IRDye® Secondaries: Recommended for Fluorescent WB

Key reasons to use secondary IRDyes for fluorescent WB include:

  • Cross-adsorption can be performed for minimal cross-reactivity between different species
  • Zero overlap between emission spectra helps avoid fluorescence across channels
  • Extensive testing is possible with over 600 proteins of different types that are optimized for fluorescent WB methodology

Researchers are advised to use the IRDye 800 (the green channel) for the detection of the weakest protein target, and the IRDye 680 (the red channel) for the strongest.

The following table shows the IRDye secondaries for various species, with the secondary antibody for each.

IRDye® Species primary was raised Secondary antibody
IRDye® 800CW Rabbit Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
Mouse Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772)
IRDye® 680RD Rabbit Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777)
Mouse Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776)

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: May 24, 2019 at 7:43 AM

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