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Fluorescent western blotting involves secondary antibodies being conjugated to fluorescent dyes and therefore circumvents the need for film and chemiluminescence.
Other advantages of fluorescent western blotting include:
Quality and performance of labeled secondary antibodies is crucial for fluorescent western blotting, the reagents must be optimized for signal:noise ratios. Furthermore, if the number of fluorescent molecules per secondary (F:P ratio) is too low the signal will be weak; if the F:P is too high, the signal will also be weak due to the inactivation of the fluorescent dye as a result of FRET.
We have generated a range of Alexa Fluor® secondary antibodies which have been manufactured and validated for fluorescent western blotting in our laboratories - ensuring quality and performance. The F:P ratios have been optimized to overcome problems highlighted above; furthermore we have developed a broad portfolio of Alexa Fluor® labeled secondary antibodies to facilitate multi-color fluorescent western blotting.
Figure legend - multi-color analysis using our Alexa Fluor® 680 (red) and 790 (green) secondary antibodies. Decreasing amounts of HeLa extract (lane 1=25µg, lane 2=20µg, lane 3=15µg, lane 4=10µg and lane 5=7µg) demonstrate staining of GAPDH (primary antibody ab83956 + anti-chicken Alexa Fluor® 790 - green, ab175787) and alpha tubulin (primary antibody 18251 + anti-rabbit Alexa Fluor® 680 - red, ab 175773).
Our range of Alexa Fluor® conjugated secondaries optimized for fluorescent western blotting.
|Alexa Fluor®||Absorption Max (nm)||Emission Max (nm)||Extinction Coefficient|
Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.