As the popularity of fluorescent western blotting grows, we thought it would be useful to provide a list of hints and tips focused on the technique.
Individual primary and secondary antibodies should be titrated - several dilutions should be tested and the optimal one which yields the highest signal to background ratio should be selected.
Fluorescent labeling of the secondary antibody under optimal conditions is crucial for performance. If the F:P (number of fluorescent molecules per secondary) is too low the signal will be weak; if the F:P ratio is too high, the signal will also be weak due to the inactivation of the fluorescent dye as a result of FRET.
Our Alexa Fluor® secondary antibodies have been manufactured and validated for fluorescent western blotting in our laboratories - thus ensuring quality and performance. The F:P ratios have also been optimized to overcome the problems highlighted above.
Enhanced photostability - choose fluorescent dyes which are resistant to photobleaching, for instance our Alexa Fluor® conjugated secondary antibodies.
Undissolved particles within buffers (milk powder in blocking buffer for example) potentially can settle on the membrane and create fluorescent artifacts. Therefore, we suggest using high quality reagents, allowing suitable time for all components to fully dissolve, and filter sterilize all buffers.
Handle the membrane with care, use blunt forceps and avoid scratching/creasing to prevent fluorescent artifacts.
Ink fluoresces, so mark the membrane with a pencil rather than pen.
Bromophenol blue itself fluoresces - so either run the dye front off the gel, or cut the gel part off, which contains the dye, prior to transfer.
Tips for multiplexing
Ensure each individual primary antibody is from a different species.
Use secondary antibodies which are highly cross-adsorbed to minimize cross species reactivity.