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

Bio-Rad
13 May 2008

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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.

Links

Bio-RadCompany website

Tags

Western BlottingWestern blotting equipment is used to transfer and identify specific proteins within a sample, reveal protein modifications, as well as give a semi-quantitative estimation of their concentration. Western blotting equipment includes all apparatus necessary to transfer proteins from gel to membrane and subsequent processing steps. Protein transfer can be performed by electroblotting with wet, semi-dry and dry transfer systems onto nitrocellulose and PVDF membranes. Blocking, washing and labeling of membranes follows, involving buffers, blocking reagents, blotting / incubation trays, labeling reagents, immunoblotting assays, antibodies and conjugates. Automated equipment for these steps is available to accelerate your lab workflow. Finally, detection and imaging of proteins can be conducted using gel documentation and imaging systems. Find the best western blotting equipment in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.ProteomicsProteomics is the systemic bioinformatics study of proteins and amino acids, including their structure, size, function and identification. Tools used in proteomics include chromatography, blotting and gels, protein arrays, mass spectrometry and ELISA and associated analysis software. Analyzers and proteomic systems should be sensitive, high resolution, fast and may be automated for high-throughput.Gel Doc / Image AnalysisGel documentation (gel doc) or gel imaging systems are used for the analysis of proteins, antibodies and nucleic acid immobilized in polyacrylamide or agarose gels, membranes or microarrays. Explore a range of a gel imaging systems, densitometers, scanners, transilluminators or UV lamp + CCD cameras for your image analysis solutions. Colorimetric, fluorescent and/or radioisotopic samples can be visualized and documented for further analysis. See gel doc / Image analysis software for quantitative 1D and 2D analysis of your samples. Find the best gel doc / image analysis products in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.Protein PurificationProtein purification is a vital step in drug discovery, therapeutics, biotech and life science research. The purification process typically involves subcellular or membrane protein extraction with cell lysis kits, separation of proteins from cell debris by filtration or spin columns, and the isolation of proteins of interest from other proteins and impurities with affinity purification (including fusion protein tags and antibody binding proteins A, G and L), immunoprecipitation or chromatographic methods, such as ion exchange, size exclusion and immobilized metal affinity chromatography. All purification methods come in multiple formats for your laboratory needs, including agarose or magnetic beads, resins, columns and filter plates. Find the best protein purification equipment in our peer-reviewed product directory: compare products, check customer reviews and receive pricing direct from manufacturers.

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