Why does choosing the right ECL matter for Western Blot?

In Western Blot analysis, a way to detect protein is an important part of the process. Chemiluminescent Western Blot has become a widely used technique as it can detect a single protein with high sensitivity, in a low amount range. However, the benefits also come with some drawbacks including a low signal-to-noise ratio, which generates high background and interferes with accurate protein quantification.

To make your blot succeed, the chosen enhanced chemiluminescent (ECL) should provide high sensitivity, less background, and a great linear range for quantification. A good ECL should minimize the amount of primary antibodies and the time you need for the processes.

Radiance from Azure Biosystem: a novel potential tool for chemiluminescent detection

         Azure Biosystem is delighted to present the new series of ECL for Western Blot analysis. Radiance, the high standard horseradish peroxidase (HRP) substrates to improve protein detection and quantification.

• Radiance ECL [AC2204]

Radiance ECL provides high sensitivity for protein detection in high femtogram range. In addition, the signal can be long lasting to 6-8 hours and it is stable at room temperature for up to 1 year, which is longer, compared to Thermo Pierce ECL, Amersham ECL, Biorad Clarity and Supersignal West Pico Plus.

• Radiance Q [AC2101]

Radiance Q provides a better ability to detect low-abundance protein, down to mid femtogram range, more sensitive than Thermo Pierce ECL Plus and Amersham ECL Prime. It is validated to be more sensitive and extended efficiency to determine the amount of protein, accurately with a wider linear range. Moreover, with its long-lasting signal, you can re-expose several times up to 10-24 hours after incubation with only 30% signal strength decay per 1 hour. Comparable efficacy compared to SuperSignal™ West Dura at a cheaper price!

• Radiance Plus [AC2103]

Radiance Plus provides the best ability to detect and quantify an extremely low-abundance protein, in the low femtograms range. With its high sensitivity and signal-to-noise ratio, you can measure the very low-abundance protein without background. The quantification of the protein could be done accurately. Furthermore, the signal duration is lasted for up to 8 hours after incubation, higher than Thermo Scientific Pierce ECL Plus. Using Radiance Plus, you can minimize the working time compared to Clarity Max Western ECL, only 2 minutes are enough for protein detection. Shelf life is also longer than Super Signal West Femto.

Why you should select Radiance as a tool for your success blot?

Radiance is the most sensitive and quantitative ECL with a versatile property that is optimized for both CCD imaging and film exposure.  You can detect low-abundance proteins in femtograms range with less background than ever. Protein can be imaged hours later after incubation or can be re-imaged several times due to its long-lasting signal. The quantification of the proteins would be accurate and easy with Radiance.

Main features of Radiance

Radiance in publications

“MARK2 associated proteins detection using Radiance ECL”

Reference: Pasapera, A. M., Heissler, S. M., Eto, M., Nishimura, Y., Fischer, R. S., Thiam, H. R., & Waterman, C. M. (2022). MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization. Current Biology.

“Autophagy proteins from HUVEC cells detection using Radiance Plus”

Reference:   Reglero-Real, N., Pérez-Gutiérrez, L., Yoshimura, A., Rolas, L., Garrido-Mesa, J., Barkaway, A., … & Nourshargh, S. (2021). Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation. Immunity, 54(9), 1989-2004.

Summary of protein detection in western blot

Choose the right ECL substrate for your Chemiluminescent Western Blot:

• Radiance ECL: an HRP substrate with a long-lasting signal and sensitivity down to low picograms. Use up to 10 times fewer antibodies than other ECL substrates.
• Radiance Q: produces a strong, long-lasting signal with extended dynamic range, perfect for detecting low and high abundance proteins within the same blot
• Radiance Plus: the most sensitive HRP substrate with attomole sensitivity and a long-lasting signal, allowing you to detect bands not visualized with other substrates.

References

Chemiluminescent and Fluorescent Westerns: Choose the Best Assay for Your Experiment. Azure Biosystem (2021). https://azurebiosystems.com/wp-content/uploads/2021/03/Chemi-vs-Fluor-app-note_1.pdf

Radiance Q Chemiluminescent Substrate. Azure Biosystem (2021). https://azurebiosystems.com/wp-content/uploads/2021/04/DC0014-005-Radiance-brochure.pdf

https://azurebiosystems.com/product-category/reagents-and-blotting-accessories/chemiluminescent-substrates/

Reglero-Real, N., Pérez-Gutiérrez, L., Yoshimura, A., Rolas, L., Garrido-Mesa, J., Barkaway, A., … & Nourshargh, S. (2021). Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation. Immunity, 54(9), 1989-2004.

Pasapera, A. M., Heissler, S. M., Eto, M., Nishimura, Y., Fischer, R. S., Thiam, H. R., & Waterman, C. M. (2022). MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization. Current Biology.