Alexa Fluor® conjugated secondary antibodies for fluorescent western blotting

by Brian Carpenter

The popularity of fluorescent western blotting is growing and presents many advantages over the traditional method of horseradish peroxidase, chemiluminescence and film. 

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:

  • Standard western blotting is semi-quantitative whereas fluorescent detection is quantitative, and covers a broad dynamic range.  For instance, during signal transduction research, quantitative analysis is important for protein expression, stability, and turnover.  All can be accurately detected and quantified using fluorescent detection.
  • Fluorescent detection facilitates simultaneous multi-color analysis on one blot, which avoids the need for stripping and re-probing.  Moreover, fluorescent detection is perfect for analyzing two different proteins which migrate at the same molecular weight.
  • The fluorescent signal is more stable than chemiluminescence. 

Quality and performance with Alexa Fluor® secondaries

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

Quantum Yield


Browse all our Alexa Fluor® products.

Alexa Fluor® is a registered trademark of Life Technologies.  Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies. 

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