Bio-Rad Technical Note Describes the Application of Multiplex Fluorescent Detection to Increase Western Blot Throughput

Bio-Rad Laboratories, Inc., a multinational manufacturer and distributor of life science research and clinical diagnostic products, announced today the availability of a technical note (tech note 5723A), which describes the application of multiplex fluorescent detection that is used to increase western blot throughput.

The most common means of analyzing protein expression levels is western blotting with detection of a single protein target. While current methods work well for studying a single target, they are unsuitable for analyzing multiple targets, particularly if the target proteins are of unknown or similar size. To analyze multiple targets, the blot is typically stripped and re-probed. However, reprobing is a time-consuming process and often some of the target protein on the blot is lost as a result of the stripping procedure.

A recent trend to resolve these issues is the application of fluorescence-based detection of proteins on western blots. This approach provides the capacity for multiplex analyses as well as greater signal stability relative to chemiluminescent detection. In addition, simultaneous probing reduces the time and labor required for western blot processing.

The advantages of fluorescent western blot detection include:

• Fast and quantitative detection of multiple proteins in a single experiment
• Comparable sensitivity to chemiluminescent detection
• Linear dynamic range — as much as 10 times greater than chemiluminescent detection
• Fewer experimental steps than chemiluminescent detection
• No substrate requirement and therefore no risk of exhausting the substrate to produce a “dead zone” in the blot
• The ability to visualize and quantitate modified forms of individual proteins, e.g. phosphorylated and total

Technical note 5723A was designed to help those who are new to fluorescent western blot detection to quickly generate reliable and reproducible results. Divided into three sections, the first section provides a complete protocol, including materials and their sources. The Tips section helps assure the success of initial experiments, and the Troubleshooting section helps provide continued success.

Availability

Technical note 5723A is available either from a local Bio-Rad sales office or can be downloaded from via the article webpage.