How to Optimize Your Chemiluminescent Western Blotts

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What is chemiluminescent detection?

The first step to troubleshooting chemiluminescent Westerns is understanding what they are. You’re in the right place! Chemiluminescence remains the most frequently used method to detect target proteins on Western blots. Many reagents are commercially available for chemiluminescent detection but all share basic characteristics.

How do you use chemiluminescence?

The secondary antibody is labeled with an enzyme, usually horseradish peroxidase (HRP). After incubation with the secondary antibody, the membrane is incubated in a solution containing a chemiluminescent HRP substrate such as luminol. When HRP reacts with the substrate, light is produced (Figure 1). Most commercial substrates also contain additional compounds that increase and stabilize the light signal, providing enhanced chemiluminescence (ECL).

chemiluminescent western blot signal
Figure 1.4. Chemiluminescent Western blotting- one signal, one protein. In chemiluminescent detection, the antigen-primary antibody complex is bound by a secondary antibody conjugated to an enzyme, such as horseradish peroxidase (HRP). The enzyme catalyzes a reaction that generates light in the presence of a luminescent substrate, and the light can be detected either by exposure to x-ray film or by a CCD-based imaging system.

Troubleshooting chemiluminescent Westerns

Because chemiluminescent detection depends on an enzymatic reaction, timing and the amount of both enzyme and substrate used have important effects on data quality. Light will only be produced while the enzyme has access to the substrate, so the blot must be imaged before the substrate is consumed and before the light signal decays. The exposure time needed to detect the signal increases as the signal declines over several minutes, leading researchers to conduct multiple exposures to try to capture the perfect image before the signal decays.

Some commercial substrates are modified to extend the lifespan of the light signal to hours rather than minutes, which can provide the researcher with more flexibility when imaging. A longer-lived signal also improves reproducibility between experiments because the signal remains constant for a longer period of time, reducing the effect of slight differences in elapsed time between substrate incubation and imaging. Now, let’s try to troubleshoot your chemiluminescent Western blot:

Why is the background on my Western blot so high? Why is there low (or no) signal?

Using too much secondary antibody can result in high background due to excess antibody binding nonspecifically to the blot. Too much secondary antibody (or too little substrate) can also reduce sensitivity because substrate will be used up too quickly and the light signal may decay before imaging can be conducted. Keep in mind that other buffer components used in washes or to dilute components can affect the reaction. Tween-20 can cause high background so should be avoided. Anything that impairs enzyme activity or alters the substrate will prevent the production of the light signal. Therefore, all buffers and reagents should be free from substances like azide that inactivate HRP, and the substrate must be protected from heat and light.

Which is better for chemiluminescent blots: film or digital imaging?

The chemiluminescent signal is usually detected either by exposing the blot to film, or by using a CCD camera. Film is expensive due to the cost of the film and of the reagents and equipment needed for developing. Importantly, film has a relatively small linear range, so the chemiluminescent signal may become saturated and it might not be possible to capture bright and dim bands with the same exposure.

How to improve your Western blots with digital imaging

Digital imagers that use a CCD camera provide a larger dynamic range, overcoming this limitation of film. Digital imaging saves time, giving instant results so researchers can quickly determine whether the selected exposure time is sufficient rather than waiting several minutes to develop film, during which time the chemiluminescent signal may be decaying. Finally, digital imaging outputs data that can be directly analyzed using densitometry to obtain quantitative information.

Additional resources for troubleshooting chemiluminescent Western blots

Check out the application note How to Improve Your Chemiluminescent Western Blots to learn more about chemiluminescent blotting. Learn more about the advantages of digital imaging of chemiluminescent Westerns in the application note Why You Should Leave the Darkroom.


Azure offers imaging systems and products to help you achieve the best chemiluminescent Westerns:

• Both the Azure Imagers and the Sapphire Biomolecular Imager include chemiluminescence imaging in addition to many other imaging modalities; find the system that best fits the needs of your laboratory

Radiance chemiluminescent substrate is designed to produce a strong, long-lasting signal for large linear dynamic range and quantitative data

• Azure Chemi Blot Blocking Buffer helps reduce background to improve signal-to-noise ratios


  1. Alegria-Schaffer A, Lodge A, Vattem K. Chapter 33. Performing and Optimizing Western Blots with an Emphasis on Chemiluminescent Detection. Methods in Enzymology. Vol 463. 2009, Elsevier Inc.

  2.  Mruk DD, Cheng CY. Enhanced chemiluminescence (ECL) for routine immunoblotting; an inexpensive alternative to commercially available kits. Spermatogenesis. 2011;1(2):121-122.


New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!

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