The advantages of fluorescence western blot detection

February 9, 2012 at 2:30 pm 1 comment

Fluorescence detection is an excellent alternative to chemiluminescence for western blot detection and offers a number of significant advantages.

  • Multiplexing — use of multiple and differently colored fluorophores for simultaneous detection of several target proteins on the same blot. When detecting multiple proteins in a fluorescent multiplex western blot, ensure the fluorescent signals generated for each protein can be differentiated. Use primary antibodies from different host species (for example, mouse and rabbit) and secondary antibodies that are cross-absorbed against other species to avoid cross-reactivity. Use fluorophores conjugated to secondary antibodies with distinct spectra so they can be optically distinguished from each other to avoid cross-channel fluorescence

  • Dynamic range — a 10-fold greater dynamic range over chemiluminescence detection and, therefore, better linearity within detection limits

  • Stability — many fluorescent molecules are stable for a long period of time, allowing blots to be stored for imaging at a later date — often weeks or months later — without significant signal loss. Most fluorescence techniques are also compatible with stripping and reprobing protocols (provided the blots are not allowed to dry out between successive western detection rounds)

A drawback of fluorescence detection is its reduced sensitivity compared to chemiluminescence methods, such that detection using low-affinity antibodies or of low-abundance proteins may yield lower signals. Photostable fluorophores, improved instrumentation, and membranes with low autofluorescence characteristics are available to allow fluorescence detection to approach the sensitivities seen with chemiluminescence techniques.

Adapted from the Protein Blotting Guide by Bio-Rad Laboratories.

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Entry filed under: Bio-Rad Tech Note, Gel Imaging and Western Blots, Miscellaneous Videos and Technotes, protein blotting, Proteomic Technotes, Proteomics, Western Blot and ELISA. Tags: , , , .

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