The popularity of fluorescent western blotting is growing and presents many advantages over the traditional method of using horseradish peroxidase, chemiluminescence and film. Fluorescent western blotting involves secondary antibodies being conjugated to fluorescent dyes, rather than enzymes, and therefore circumvents the need for film and chemiluminescence.
Other advantages of fluorescent western blotting:
- 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, as a result blots can be stored and imaged at a later date.
Quality and performance with Alexa Fluor® 680/790 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 (under labeled) the signal will be weak; if the F:P is too high (over labeled), the signal will also be weak due to the inactivation of the fluorescent dye as a result of Förster resonance energy transfer (FRET).
We have generated a range of Alexa Fluor® 680/790 secondary antibodies which have been manufactured and validated for fluorescent western blotting in Abcam’s 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® 680/790 labeled secondary antibodies to facilitate multi-color fluorescent western blotting.
- Due to minimal-spectral overlap of the Alexa Fluor® 680 and 790, two color analysis is even easier.
- To ensure quality and performance – each individual product within the range is validated in fluorescent western blotting, and for clarity, the corresponding QC data published.
To learn more, please visit, www.abcam.com/fluorescent-wb
Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.