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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.
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.