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Increased Western Blot Throughput with Multiplex Fluorescent Detection
Sponsored,vendor-submitted protocol   Sponsored by Bio-Rad Laboratories    Published in November 2008 (p.39) DOI: 10.2144/000113013

The most common method for analyzing protein expression levels is Western blotting with detection of a single specific protein target, using horseradish peroxidase– or alkaline phosphatase–conjugated antibody probes combined with colorimetric or chemilumines-cent detection. However, multiplex analyses using these techniques are time-consuming and imprecise. Fluorescence-based detection of Western blots is becoming a popular way to overcome these drawbacks. This approach provides the capacity for multiplex analyses and simultaneous probing and thus reduces the time and labor required for Western blot processing. Also, less starting sample is required, since only one blot has to be prepared to analyze multiple proteins. Using a combination of two antibodies selected for minimal cross-reactivity, fluorescent detection methods enable simultaneous quantitative analysis of two or more proteins and normalization of the signal to an internal protein standard to allow measurement of protein expression levels directly from a blot.


The numerous advantages of fluorescent Western blot detection include:

  • Fast and quantitative detection of multiple proteins in a single experiment.
  • Sensitivity comparable to that of chemiluminescent detection.
  • Linear dynamic range up to 10 times greater than that of chemiluminescent detection.
  • Fewer steps than chemiluminescent detection.
  • No substrate requirement, therefore no danger of exhausting the substrate and producing a dead zone in the blot.
  • The ability to visualize and quantitate both phosphorylated and nonphosphorylated forms of individual proteins.
  • Ability to use the Bio-Rad Precision Plus Protein™ WesternC™ standards to estimate molecular weight directly from fluorescent multiplexed blots.


A full protocol is available for using fluorescent Western blot detection to quickly generate reliable and reproducible results. The first section provides a complete protocol, including materials and their sources. The Tips section will help assure the success of initial experiments, and the Troubleshooting section will help provide continued success.


Address correspondence to Bio-Rad Laboratories, Inc., Laboratory Separations Division, Life Science Group; Tel.: 1-800-4BIORAD;

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