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Fluorescence-based imaging has grown to be a popular choice for Western blot detection and analysis, owing to its ultra-sensitive,
flexible and convenient capabilities. The FluorChem R Imaging System is compatible with far-red and near-infrared (NIR) dyes and is designed to acquire high-quality
images from fluorescently labeled, multicolor Western blots.
In this application note, we’ll give you tips on how to image Proteome Profiler Antibody Arrays using chemiluminescent or IR detection on FluorChem M or FluorChem R systems, and show the results from a couple of arrays.
Total protein normalization is the preferred method when it comes to normalizing Western blot protein signals, as it simplifies the quantitation workflow and eliminates the need for a housekeeping protein to normalize against. This method also avoids situations where the housekeeping protein expression changes between samples or is expressed in levels outside the dynamic linear range of your protein of interest. You also eliminate the need to strip and re-probe the membrane if your housekeeping gene migrates too close to your protein of interest.
Stain-Free™ gel technology simplifies the process further by removing the need for detecting total protein amounts after protein separation with a colorimetric or fluorescent stain. Stain-Free gels (Bio-Rad) contain a trihalo compound that crosslinks with tryptophan residues in proteins, producing a fluorescent signal under UV light. The signal can be detected right after separation in the gel and after transfer to a membrane.
In this application note, we demonstrate how to combine the ease-of-use of FluorChem™ imaging systems with StainFree gels to normalize the chemiluminescent signal from Western blots using AlphaView® software.
Western blotting is an essential tool in protein research. Traditionally, proteins are labeled with secondary antibodies tagged with chemiluminescent substrates and are detected using photographic film. Chemiluminescent Western blotting has been a dominant method of detection for identification of proteins in cell or tissue samples for decades. While it offers significant advantages over other methods it also has certain limitations, one being the ability to analyze multiple proteins on the same blot.
Fluorescent imaging technology gives researchers the ability to move beyond the traditional Western blot assay by offering the flexibility to multiplex and detect co-migrating proteins. Native and phosphorylated isoforms can be probed simultaneously and quantified without the additional time and signal loss in sequential stripping and re-probing.
Simple Imaging Analysis - Digital Darkroom
Get the right picture, right away with the protocol-driven touchscreen interface only available on Simple Imagers running Digital Darkroom.
Protein analysis plays an important role in stem cell research, and for most researchers, Western blotting is a key component of their toolbox. From verifying pluripotency and identifying lineage-specific cell types to identifying key modulators of cell signaling pathways, to evaluating disease models and therapeutic approaches, the right protein analysis
techniques can help you achieve success. In this eBook, we examine technological advances in Western blotting and protein analysis that are at the forefront of stem cell research. Learn how automated Western systems and Single-Cell Westerns can advance your stem cell research.
Research into prevention and treatment of disorders of the brain and nervous system are in greater demand as the world’s population expands. This group of diseases result in more hospitalizations than any other group, including heart disease and cancer. Odds are that you know someone affected by a neurological disorder, as The World Health Organization estimates that one billion people are affected by them, contributing to 11% of the world’s disease burden.
The FluorChem Q gives me clean and publication-quality results. This has helped me enormously todo accurate protein analysis which is very critical for preclinical validation of drugs.
Picking the appropriate detection method for your Western blot
In this technical note, we discuss the features and terminology of CCD technology relevant to biological imaging, enabling you to understand how the components of the entire system can contribute to performance.