Azure c600 Gel Imaging System

This application note demonstrates how sensitive high-resolution imaging systems, such as the Azure c600, can quickly and easily image fluorescently tagged NDV in chicken embryos, and, to some extent, quantify the viral load in various embryonic tissues.

This application note demonstrates a procedure to obtain three-color fluorescent blots using WesternDot® reagents. Multicolor detection is a powerful application of fluorescent Western blotting. Two or more proteins can be detected on one blot using antibodies labeled with different fluorophores. The ability to simultaneously assay multiple proteins on a Western blot represents a significant advantage of fluorescent detection over chemiluminescent detection, because chemiluminescent detection requires stripping and re-probing a blot or using duplicate blots to detect two proteins, approaches that can introduce errors and variation.

The Azure cSeries imagers are easy-to-use and reliable instruments for Western blot imaging, and enable labs to use one system for fluorescent and chemiluminescent Westerns. The simple user interface allows fast imaging of all sample types. Full upgradability means customers can have confidence that their system will grow and adapt with their needs. Customers no longer have to choose one chemistry for their Western blots; they can buy one system and customize it to their needs.

When most people think of imaging their Western blot, they think of going into a darkroom and exposing the blot to film to detect a chemiluminescent signal. The advent of digital imaging has transformed Western blot detection. This paper outlines how Western blotting has changed, and reviews the qualities to look for in a digital imager.

Chemiluminescent Westerns are popular assays for assessing protein expression. In this indirect detection method, chemiluminescent substrates emit light when reacted with an antibody conjugated to an enzyme. The emitted light is then captured and recorded. It is a highly sensitive assay and can detect femtograms of protein. Although it is only semi-quantitative, it is useful for detecting the presence or absence of a protein. Here are some tips to improve your chemiluminescent Western blots.

To perform a Western blot, proteins are separated according to molecular weight by electrophoresis through a polyacrylamide gel. The proteins are then transferred from the gel onto a solid membrane support for subsequent immunodetection by protein-specific antibodies. Transfers can be performed by wet transfer or under semi-dry conditions. This application note outlines the benefits and drawbacks of each system which must be considered when optimizing transfer conditions.

Fluorescent Western blots are the gold standard for quantitative Westerns. They are ideal for detecting multiple proteins simultaneously (multiplexing), allowing in-lane normalization and detection of your protein of interest and loading control at the same time. In addition, post-translational modifications can be studied and quantitated easily. This application note suggests how to improve your fluorescent Western blots.

This application note evaluates chemiluminescent and fluorescent western blotting to enable you to choose the best assay for your experiment. Learn how fluorescent detection can complement, enhance and provide new insight when coupled with chemiluminescent detection.

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