chemiluminescent western Blots

Chemiluminescent Westerns 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). 

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.

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.

Enzymes used for chemiluminescent detection

Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are two commonly used enzymes for chemiluminescent detection. The sensitivity of chemiluminescent detection depends on the choice of substrate. 

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 (see Table below).

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

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?” 

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. 

 

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

 

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.

Furthermore, 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 (Figure 3).

Ready to roll up your sleeves? Learn how to image a chemiluminescent Western blot from start to finish with this chemiluminescent Western blot protocol.

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