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 http://www.bio-rad.com.
About Bio-Rad
Bio-Rad Laboratories, Inc. (AMEX: BIO and BIOb) has remained at the center of scientific discovery for more than 50 years manufacturing and distributing a broad range of products for the life science research and clinical diagnostic markets. The company is renowned worldwide among hospitals, universities, major research institutions, as well as biotechnology and pharmaceutical companies for its commitment to quality and customer service. Founded in 1952, Bio-Rad is headquartered in Hercules, California, and serves more than 85,000 research and industry customers worldwide through its global network of operations. The company employs approximately 6,300 people globally and had revenues approaching $1.5 billion in 2007. For more information, visit www.bio-rad.com.
For more information contact: Charles Martin Bio-Rad Laboratories, Inc. 510-741-5286 Charles_Martin@bio-rad.com
