Fluorescent Western Blotting

Fluorescent western blotting is a method that is increasing in popularity because it addresses the need for accurate, quantitative determination of protein expression. The basic principle of the technique is that the level of fluorescence emitted by a fluorophore conjugated to the detection antibody is directly related to the level of protein expression. Unlike traditional western blotting detection methods, fluorescence is not subject to enzyme kinetics; making quantitation more reproducible. Furthermore, method development is simplified due to the linearity of the reaction. An additional benefit of the chemistry is blots can be cataloged for extended periods of time when stored properly.

By combining different antibody species with fluorophores of varying wavelengths, you can use fluorescent western blotting to create multiplex experiments. This will save you time and sample and allow you to answer more complex questions. Multiplexing also allows you to perform accurate analysis when there are only small shifts in molecular weight, e.g., post-translational modifications. By optimizing your experiments to include fluorescent multiplexing, you can avoid the errors commonly associated with stripping, reprobing, and cutting blots and ensure you are publishing quantifiable, reproducible results.