- 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.
- Membrances have a tendency to autofluoresce resulting in high background. Choose specialist membranes which have been engineered to overcome this issue - low fluorescence western membrane (PVDF).
- 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.
- Choose secondary antibody conjugates with optically distinct spectra, for instance Alexa Fluor® 680 and Alexa Fluor® 790 to avoid cross channel fluorescence.
- Optimize the conditions for the individual antibody pairs (primary + conjugated secondary) separately, before attempting multi-color analysis.
Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.