chemiluminescent western Blots

Table of Contents

What are 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).

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.

What do chemiluminescent Western blots tell you?

Chemiluminescence is a popular indirect detection method for Western blotting. Chemiluminescent Western blots are 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?”.

How does chemiluminescent imaging work?

Imaging of a chemiluminescent Western blot is done via exposure of the blot to X-ray film; however, imaging of a chemiluminescent Western blot can also be done using a CCD-based imaging system.

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

Download a free Chemiluminescent Western blotting protocol

Strengths and weakness of chemiluminescent Western blots

There are certain strengths and weaknesses for chemiluminescent Western blotting that you should be aware of: including sensitivity and compatibility with either film or digital detection for imaging (Table 1).

Table 1. Advantages and disadvantages of chemiluminescent Western blotting

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

Why chemiluminescence is used in Western blotting

Chemiluminescent detection is often used for Western blots 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 this question:

  • Is my protein present or not?

Chemiluminescent detection is not very good at addressing these questions:

  • 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?

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. 

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 How to Improve Chemiluminescent Western Blots to learn more about chemiluminescent Western blotting.

Enzymes used for chemiluminescent detection

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.

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.

Chemiluminescent Western Blot imaged with Azure Imager
Figure 2. Chemilumescent Western blot detected using film. Processed in a darkroom.

What substrates are best for detection of chemiluminescent Western blots?

There are many available options when it comes to chemiluminescent Western blotting substrates . Check out Table 1 to figure out which chemiluminescent substrate is best for you, based on the target protein of interest’s abundance and sensitivity level.

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.

Table 2. Radiance HRLP chemiluminescent substrate comparison table

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

Request a free sample to try out a new chemiluminescent HRP substrate to see which is best for you.

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. While X-ray film serves to furnish qualitative and semi-quantitative data and is valuable for verifying the existence of target proteins, modern digital imaging systems utilizing cooled CCD cameras present numerous benefits, including swift data acquisition, quantitative analysis, superior resolution, heightened sensitivity, along with a broader dynamic range, when compared to film. If you’re looking for a new chemiluminescent Western blot imager, check out this blog post for five features you need to look for in your lab’s next big purchase.

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 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; the Azure Imaging Systems, even offer built-in auto-detection, Auto-Illumination and Filter Control, and pre-calibrated focus. These features make 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 for chemiluminescent Westerns over traditional darkroom practices, read Why You Should Leave the Darkroom.

Are there imagers that only detect chemiluminescence?

If your application needs stop at chemiluminescence, chemiSOLO may be the right option for you. It is a portable, personal Western blot imager now available in the United States and worldwide. It’s the first chemiluminescent imager on the market that can be controlled by a table, smartphone, or laptop– without the need for additional software downloads.

In addition to Western blots, 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 it to be controlled by phone, tablet, or PC, without the need to install any additional software.

Whatever your Western blot imaging needs are, let the  Western blotting experts help at Azure Biosystems! If you’re unsure of which system you need for chemiluminescent Western blotting, an 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.

Systems for Chemiluminescent Western Blotting

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

Azure 280

Basic imaging system for chemiluminescent Western blots and DNA gels

Reagents Needed


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!

Resources for Chemiluminescent Western Blotting

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