Benefits of fluorescent detection for quantitative Western blotting.

Abstract

Western blotting is a well‐established and widely used technique to confirm the identity and presence of proteins from a variety of sources. The development of highly sensitive detection reagents together with advanced imaging techniques has made Western blotting a potential tool for quantitative protein analysis. A prerequisite for obtaining quantitative Western blotting data is that the signal response is proportional to the amount of protein. Fluorescent Western blotting detection is a direct detection method where the secondary antibody is conjugated to a fluorophore. The obtained fluorescent signals are stable and directly proportional to the protein amount. Fluorescent detection systems are typically characterized by their high sensitivity and broad linear dynamic range, and are as such well adapted to quantitative Western blotting. Moreover, fluorescence detection enables simultaneous detection of more than one protein on the same blot (multiplexing), even when the proteins have similar size, by utilizing secondary antibodies conjugated to fluorophores that are spectrally differentiated from each other. This makes fluorescent Western blotting a useful tool for accurate monitoring of changes in protein abundance and for detection of posttranslational modifications.Here we demonstrate the benefits of fluorescent detection in quantitative Western blotting using Amersham™ ECL Plex™ and Image Quant™ LAS 4010 CCD camera. Proteins are identified and expression levels determined using a multiplexed Western blotting approach..