The Advantages of Fluorescent Western Blotting

Our antibody databaseincludes many thousands of proteins, and it is constantly being enriched. Modern developments mean that, whereas scientists would once have searched for one protein in a single sample, now they search for several – often simultaneously, and in minute quantities.

Western blotting is a standard antibodyassay technique, which for many years was governed by chemoluminescent signalling. This relies on a single light signal. In recent years, this has given way to fluorescence, which has a broad palette of fluorescent colors and thus can be used to screen several proteins simultaneously, quantifying their presence by means of a control.

An advance on this has been the development of phosphospecific antibodies, which when used alongside standard antibodiescan determine the fraction of phosphorylated to non-phosphorylated protein in a sample. Previously, the only way to do this was to strip and re-probe the blot, owing to the closeness of the two isoforms.

Fluorescent signalling also has the advantage of quantitative reliability. The intensity of chemoluminescense varies depending on the length of time the substrate is incubated, the freshness of the reagents and variations in temperature, making it difficult to compare samples or repeat experiments accurately. With fluorescent signalling, these problems don’t arise. Generally, the light signal output is comparable to the amount of protein present.

We at Novus Biologicals have an extensive antibody database, with reagents that are prepared to an incredibly high level of purity and sensitivity. Combined with modern fluorescent imaging techniques, they are making a sizable contribution to our understanding of molecular and cellular biology, and disease.