How to Optimize Your Chemiluminescent Western Blots

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Chemiluminescent detection depends on an enzymatic reaction so 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.

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. Anything that impairs enzyme activity or alters the substrate will prevent the production of the light signal. Avoid using Tween-20, as it can cause high background. Instead, use Azure Chemi Blot Blocking Buffer to help reduce background and improve signal-to-noise ratios on your Western blot.

chemiluminescent western blot signal
Figure 1. Chemiluminescent Western blotting- one signal, one protein.

Best substrate to use for chemiluminescent Western blots

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. That’s where Radiance comes in. Radiance is a specially formulated, chemiluminescent substrate designed to produce a strong, long-lasting signal for large linear dynamic range and quantitative data.

A longer-lived signal 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.

  • Azure Quick Tip: All buffers and reagents should be free from substances like azide that inactivate HRP. The substrate must be protected from heat and light.

Is film or digital imaging better for chemiluminescent Western blots?

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. Film has a relatively small linear range, so the chemiluminescent signal may become saturated. It might not be possible to capture bright and dim bands with the same exposure.

LEARN MORE: Check out this application note How to Improve Your Chemiluminescent Western Blots to learn more about chemiluminescent Western blotting. If you want to learn more about the advantages of digital imaging of chemiluminescent Westerns read Why You Should Leave the Darkroom.

Connecting laptop to Azure chemiSOLO chemiluminescent imager
Azure chemiSOLO is a personal Western blot imager used to capture pictures of colorimetric blots or visible-stained protein gels, like Coomassie blue or silver stain. A unique web browser interface allows the chemiSOLO to be controlled by phone, tablet, or PC, without the need to install any additional software.

Digital imagers for the best chemiluminescent Western blots

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. Digital imaging outputs data that can be directly analyzed using densitometry to obtain quantitative information.

Looking for an affordable option to quickly image Western blots? The Azure chemiSOLO is a newly launched, personal Western blot imager that’s capable of detecting low-expressing proteins with femtogram sensitivity. chemiSOLO also captures marker images at the push of a button. Learn more about chemiSOLO by clicking here.

Azure Imagers also allow you to use multiple binning options to collect more light. Both the Azure Imagers and the Sapphire Biomolecular Imager include chemiluminescent imaging, in addition to many other imaging modalities; find the system that best fits the needs of your lab by clicking below.

What is chemiluminescent detection?

With chemiluminescent detection, a primary antibody binds to the target protein on a membrane, and the location of the primary antibody is detected using a secondary antibody conjugated to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP).

A substrate for the enzyme is added and when the enzyme acts on the substrate, light is emitted (Figure 1). The light can be detected using an imager with a CCD camera or x-ray film. The sensitivity of detection depends on the choice of substrate—commercially available substrates for HRP can detect proteins in the femtogram range.

How do you use chemiluminescence to detect proteins?

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. 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).

SOURCES

  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.

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