chemiluminescent western Blots

Chemiluminescent Western blots are popular assays for assessing protein expression. In this indirect detection method, chemiluminescent substrates emit light when reacted with an antibody conjugated to an enzyme. (Figure 1).

Chemiluminescence is a popular indirect detection method for Western blottingThis technique is very good at answering the question, “Is my protein there or not?” and can provide a qualitative answer to the question “Is the amount of my protein different between these two samples?”

In chemiluminescent detection, the primary antibody binds to the target protein on the 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). This reaction is shown in Figure 1.

Depiction of chemiluminescent Western blot signal
Figure 1. 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, like the Azure 300.

How does chemiluminescence imaging work?

Western blot imaging of a chemiluminescent Western blot is done via exposure of the blot to x-ray film and can also be done using a CCD-based imaging system.

Chemiluminescent detection is often used because it is specific, easy to perform, and highly sensitive—proteins can be detected at femtogram levels (Table 1).

Chemiluminescent Western blotting protocol

Compatible with film or digital imagingSignal dependent on exyme kinetics
Easy, familiar chemistrySingle protein only, loading controls require stripping and reprobing

Table 1. Advantages and disadvantages of chemiluminescent Western blotting

Why is chemiluminescence used in Western blotting?

Chemiluminescent detection is often used because it is specific, easy to perform, and highly sensitive. The labeled secondary antibody can be used across multiple experiments to detect any primary antibody of the correct species, making the approach cost-effective.

Chemiluminescent detection is very good at answering the question, “Is my protein there or not?” However, chemiluminescent detection is not very good at addressing questions such as, “How much of my protein is present relative to another protein? How much of my protein is in one sample compared to another sample? How do I control for sample loading inconsistencies?” 


Chemiluminescent Western Blot imaged with Azure Imager
Figure 2. Chemilumescent Western blot

Unlike fluorescent tags, where one or more different proteins can be probed simultaneously using antibodies labeled with spectrally distinct fluorophores, chemiluminescent reactions emit light over a broad range of wavelengths. Thus, with chemiluminescent detection, emission wavelengths cannot be used to distinguish signals from different proteins. Instead, the proteins must be well-resolved electrophoretically. 

Enzymes used for chemiluminescent detection

HRP and AP are two commonly used enzymes for chemiluminescent detection. The sensitivity of chemiluminescent detection depends on the choice of substrate.

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 a CCD camera or x-ray film (Figure 2). The sensitivity of detection depends on the choice of substrate—commercially available substrates for HRP can detect proteins in the femtogram range. Check out Table 2 to see which HRP chemiluminescent substrate is appropriate based on your abundant proteins of interest.

SubstrateDetectsSignal DurationSignal StrengthSensitivity
Radiance ECLMedium to high abundance proteinsLow pg to high fg+++6 to 8 hr
Radiance QLow to medium abundance proteins10 to 24 hr++++Mid fg
Radiance PlusLow to very low abundance proteins6 to 8 hr+++++Low fg

Table 2. Radiance HRLP chemiluminescent substrate comparison table

Radiance Plus is the most sensitive HRP substrate available for chemiluminescent Western blotting from Azure Biosystems. With attomole sensitivity and a long-lasting signal, Radiance Plus allows you to detect bands not visualized with other substrates.

High signal-to-noise and a large dynamic range make it ideal for quantifying low-intensity bands. Radiance ECL is an HRP substrate with long-lasting signal and sensitivity down to low picograms. Users are able to use up to 10 times less antibody when compared to other substrates on the market.

Radiance Q is an HRP substrate that provides a strong, long-lasting signal, the broadest useful linear range, and high sensitivity for the most quantitative chemiluminescent Western assays. Request a free sample to try out any of Azure’s sensitive chemiluminescent HRP substrates to see which is best for you.

For example, proteins with small differences in molecular weight, such as the same protein with and without a posttranslational modification, tend to co-migrate during electrophoresis making them difficult to visualize simultaneously using chemiluminescence since the bands will most likely overlap. Overlapping bands can also impact detection of normalization and loading controls. Unless these controls are well resolved electrophoretically from the protein-of-interest, the blot must be either stripped and reprobed to detect the control, which renders the blot non-quantitative, or the controls must be placed on a separate blot, which is not a true loading control.

Because chemiluminescence relies on an enzyme-substrate reaction, the amount of signal (emitted light) is subject to variations in reaction kinetics, which can be affected by reaction conditions, i.e. pH, temperature, substrate concentration, and enzyme concentration. This inherent variability makes chemiluminescence, at best, a semi-quantitative detection chemistry.

The traditional use of x-ray film as a method of visualization suffers from dynamic range limitations of the film that can often lead to signal saturation. Using a digital imager can increase the linear dynamic range, allowing easier detection of low abundance proteins while limiting saturation when detecting high abundance proteins.

LEARN MORE: Check out this application note How to Improve Your Chemiluminescent Western Blots to learn more about chemiluminescent Western blotting.

Choosing a chemiluminescent Western blot imager

Today, Western blot detection is no longer limited to the capabilities of a x-ray film in a darkroom. There are many digital Western blot imaging systems available on the market that allow for accurate quantitation of a range of signal intensities in Western blotting. Both chemiluminescent and fluorescent detection can be documented with a CCD-camera-based Western blotting system, like the Azure Imaging Systems (the only imager on the market to use lasers for higher signal), or through photodiode or PMT detection with the new Sapphire FL Biomolecular Imager.

Azure imaging systems offer:

  • Quantitative chemiluminescent imaging

  • Speed and sensitivity of X-ray film

  • High-resolution imaging perfect for publications
  • IQOQ Validation

A great benefit to using digital imaging systems is the ability to adjust the exposure time; some systems, like the Azure Imaging Systems, even offer built-in auto-detection, Auto-Illumination and Filter Control, and pre-calibrated focus, which makes capturing images easy and cuts down on time. An integrated touchscreen also allows ease of use and can easily be controlled by an external PC, if required.

LEARN MORE: If you want to learn more about the advantages of digital imaging of chemiluminescent Westerns read Why You Should Leave the Darkroom.

Learn more about the Azure Imaging Systems, including full product specifications, user manuals and resources.

If your application needs stop at chemiluminescence, the new Azure chemiSOLO is now available in the United States and worldwide. It can easily and quickly image chemiluminescent Western blots by using your phone, tablet, or laptop- without the need for additional software downloads. It’s the first personal Western blot imager of its kind on the market.  In addition to Western blots, the chemiSOLO captures pictures of colorimetric blots or visible stained protein gels, like Coomassie blue or silver stain.

Azure chemiSOLO connecting to an external laptop
chemiSOLO is a new personal chemiluminescent Western blot imager that detects low-expressing proteins with femtogram sensitivity and captures marker images at the push of a button. A unique web browser interface allows the Azure chemiSOLO to be controlled by phone, tablet, or PC, without the need to install any additional software.

Whatever your Western blot imaging needs are, let our Western blotting experts help! If you’re unsure of which system you need for chemiluminescent Western blotting, an Azure Biosystems expert is available to help you choose the right option. Fill out the form on this page and let us know what you’re looking for in a system. That’s it. We’ll match you up with the best fit.


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!
Products for Western blotting

A portable, personal chemiluminescent Western blot imager controlled by mobile device

Basic imaging system for chemiluminescent Western blots and DNA gels

Resources for Chemiluminescent Western Blotting

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