Fluorescent Western blotting uses secondary antibodies directly conjugated to fluorescent dyes. Fluorophores for fluorescent Western blotting can be chosen based on their specific excitation and emission spectra, enabling multiplexing (detection of multiple proteins simultaneously, Figure 1) for faster, more efficient studies. One of the biggest advantages of fluorescent detection versus chemiluminescence is the ability to use more than one antibody per assay (see the Table).
With multiplexing, normalization and loading controls can be imaged at the same time and on the same blot as the sample. In addition, the ability to use different fluorophores enables visualization of proteins that are not well-separated electrophoretically, for more convenient imaging of the same protein with and without post-translational modifications.
|Multiplex capability||Can be less sensitive than chemiluminescence|
|Increased quantitative accuracy||Membranes' auto fluorescence can increase background|
|Fuorescent label stability allows blots to be|
stored and re-imaged later
Fluorescent Western blots are the gold standard for quantitative Westerns. Chemiluminescent Westerns are limited by the variable kinetics of the enzyme-substrate reaction; however, the amount of light emitted from fluorophores is highly consistent. The amount of light is directly proportional to the amount of protein on the membrane. This consistency means fluorescent detection can provide a truly quantitative analysis of the proteins in question.
Fluorescent Westerns are typically visualized using a digital imager, rather than X-ray film (Figure 2). The newer generation of imaging systems often contain sophisticated cameras that exhibit a broader dynamic range than film, thus avoiding the signal saturation problems that limit the dynamic range of film. Finally, fluorescent dyes are relatively stable; blots can be archived and imaged months after the initial experiment as long as precautions are taken to avoid photo-bleaching of the fluorophores.
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Western blotting is an important tool for researchers studying signal transduction and other processes where understanding the phosphorylation state of a protein is important for