Chemiluminescent substrates are popular because they offer several advantages over other detection methods. These advantages have allowed chemiluminescence to become the detection method of choice in most protein laboratories. Using chemiluminescence allows multiple film exposures to be made, which enables optimization of signal to noise. The detection reagents can be removed and the entire blot reprobed to visualize another protein or to optimize detection of the first protein. A large linear response range allows detection and quantitation for a large range of protein concentrations. Most importantly, chemiluminescence yields the greatest sensitivity of any available detection method.


In contrast to fluorescence western blotting systems, chemiluminescent detection occurs when energy from a chemical reaction is released in the form of light. The most popular chemiluminescent western blotting substrates are luminol-based. For example, in the presence of horseradish peroxidase (HRP) and peroxide buffer, luminol oxidizes and forms an excited state product that emits light as it decays to the ground state. Light emission occurs only during the enzyme-substrate reaction; therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases. The two most common enzyme reporters that catalyze chemiluminescent reactions are HRP and alkaline phosphatase (AP). Enzyme-conjugated antibodies are used for western blotting, and light-producing reactions are captured with X-ray film. However, charge-coupled device (CCD) camera–based digital imaging instruments have been used for more convenient and versatile data capture. 

The choice of substrate is determined by the reporter enzyme that selected. Specifically, luminol- and acridan-based reagents are chemiluminescent HRP substrates. For chemiluminescent detection of AP, acridan- and 1,2-dioxetane-based substrates are available. Substrates are the limiting reagent of the reaction, and light signal diminishes as the substrate is consumed. With well optimized experiments using the correct concentration of enzyme-conjugated antibody, light output remains stable for several hours, enabling a high degree of sensitivity in protein detection.

Watch this video on western blot detection using chemiluminescent substrates

Chemiluminescent reaction of luminol. Chemiluminescence is a property of chemical reactions that emit light as a byproduct. Luminol is one of the most widely used chemiluminescent reagents. The oxidation of luminol by peroxide results in creation of an excited state product called 3-aminophthalate. This product decays to a lower energy state by releasing photons of light.

Light output generated during a chemiluminescent luminol reaction is relatively short lived; therefore, enhanced chemiluminescent (ECL) substrates have been developed. For example, use of an enhancer with luminol increases signal sensitivity, intensity, and duration of the enzyme-substrate reaction.  Thermo Scientific Pierce ECL substrate is appropriate for western blot applications in which abundant proteins are being probed or where the experiment has been optimized. However, substrates providing highly sensitive protein detection have been developed. For example, Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate enables picogram- to high femtogram–level protein detection by western blot analysis.

Luminol-based Enhanced Chemiluminescent (ECL) western blotting substrate. β-actin and β-galactosidase protein in HeLa cell and E. coli lysates, respectively, were detected by western blotting. The membranes were blocked with 5% nonfat milk and probed with primary antibody at 1μg/mL. The membranes were washed, then incubated with 0.2μg/mL of HRP-conjugated goat anti-mouse IgG and washed again. Working solutions of the Thermo Scientific Pierce ECL Western Blotting Substrate were prepared according to the manufacturer's instructions and added to replicate membranes for one minute. The membranes were removed from the substrates and placed in plastic sheet protectors and exposed to Thermo Scientific CL-XPosure Film and developed.

Protein Detection Technical Handbook

This 84-page handbook provides a comprehensive look at the last step in the western blot workflow—protein detection. With a variety of detection techniques to choose from, including chemiluminescence, fluorescence, or chromogenic detection, performers of western blot analysis can select a technology that matches experimental requirements and available instruments. For quick visualization or precise quantitation, single-probe detection or multiplexing, Thermo Fisher Scientific offers a range of reagents and kits for western blot protein detection and subsequent analysis.

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Data capture for chemiluminescence

Chemiluminescent western blot signal can be captured with X-ray film, charge-coupled device (CCD) camera–based digital imaging instruments, and phosphorimagers that detect chemiluminescence. Although X-ray film provides qualitative and semi-quantitative data and is useful to confirm the presence of target proteins, CCD camera–based imaging instruments offer the advantages of qualitative analysis, instant image manipulation, higher sensitivity, greater resolution and a larger dynamic range than film. Additionally, there is no need for a darkroom.

Although electronic data capture with CCD-based imaging instruments is growing in popularity as the technologies improve and instrument prices decline, most of the data obtained from western blotting with chemiluminescence is still captured on film. Often, it is necessary to expose several films for different time periods to obtain the proper balance between signal and background. The goal is to time the exposure of the membranes to the film so that the desired signal is clearly visible while the background remains low. This is difficult to accomplish since the process cannot be observed and stopped when the desired endpoint is reached. If the film is not exposed long enough (underexposed), the signal will not be visible. If the film is exposed too long (overexposed), the signal may be lost in the background or separate bands may become blurred together.

Overexposed film can be optimized after exposure by using reagents formulated to effectively reduce the film exposure time without altering the integrity of the data. This is done at the lab bench while watching the film, and the process can be halted when the signal is clearly visible and background is at a minimum.

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Molecular weight markers for chemiluminescent detection

For detection of any western blot, it is desirable to use prestained molecular weight markers that are transferred to the membrane along with the protein sample. The appearance of the molecular weight markers on the membrane allows estimation of molecular weights for any protein bands that are detected as well as effective separation of the proteins of interest in the gel prior to the transfer step. When chemiluminescent detection is used for western blotting, protein bands are detected on film or with digital imaging equipment. Unless modified, molecular weight markers do not show up on film or the imaging system since they do not produce an output of light. For this, there are several solutions described below.

Use of SuperSignal molecular weight marker for chemiluminescent western blot detection. Western blot analysis of HA-epitope tag was performed using samples from Thermo Scientific Pierce HA-Tag IP/Co-IP Kit with Thermo Scientific SuperSignal Molecular Weight Protein Ladder in Lane 1, an HA-tagged positive control lysate in Lane 2, unbound flow-through in Lanes 3 and 4, and two elutions from the immobilized anti-HA resin in Lanes 5 and 6. The membrane was probed with an HA Tag Monoclonal Antibody (Cat. No. 26183) at a dilution of 0.1 ug/mL followed by chemiluminescent detection with Thermo Scientific SuperSignal West Dura Extended Duration Substrate (Cat. No. 34075).

One solution is the use of molecular weight markers that employ antibody binding domains from Protein A or G. The antibody capture domains of these proteins are engineered into the molecular weight markers and thus bind to the antibodies used in the western blot, allowing a signal to be generated and captured alongside the experimental data. The application of these markers is limited because the variable affinities of antibodies for Protein A and G result in variable levels of signal. Many antibodies, such as those of the mouse IgG1 subclass, do not bind strongly to Protein A or G.

Another popular option is to use biotinylated protein molecular weight markers, which allow the use of streptavidin alongside the detection antibodies or when biotinylated primary detection antibodies are used.

Recommended reading

  1. G.H. Thorpe, L.J. Kricka. (1986) Enhanced chemiluminescent reactions catalyzed by horseradish peroxidase. Methods in Enzymology 133:331–54.
  2. Alegria-Schaffer, A., et al. (2009) Performing and optimizing Western blots with an emphasis on chemiluminescent detection. Methods Enzymol 463:573–99.
  3. Gassmann, M., Granacher, B., Rohde, B. and Vogel, J. (2009) Quantifying Western blots: Pitfalls of densitometry. Electrophoresis 30:1845–55.