Western blotting is a powerful technique that enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. In a conventional western blot, protein samples are first resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to the membrane. Following a blocking step, the membrane is probed with a primary antibody (polyclonal or monoclonal) that was raised against the antigen in question. After a subsequent washing step, the membrane is incubated with a secondary antibody conjugated to an enzyme that is reactive toward the primary antibody. The two most common enzyme reporters that catalyze chemiluminescent reactions required for generating a recordable signal are horseradish peroxidase (HRP) and alkaline phosphatase (AP).
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 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. Both HRP and 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.
Watch this video on western blot detection using chemiluminescent substrates
Chemiluminescent western blot detection methods
The choice of substrate for chemiluminescent western blotting is determined by the reporter enzyme that is 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.
Figure 1. 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.
As HRP is the most popular enzyme used in western blotting it will be discussed throughout this document as our example. Several varieties of Thermo Scientific Pierce ECL and SuperSignal chemiluminescent HRP substrates are available that provide different levels of sensitivity for chemiluminescence western blotting. Refer to Table 1 to identify the most appropriate HRP chemiluminescent substrate based on the abundance of your target protein of interest, abundance of sample containing the target protein, and the level of sensitivity and type of instrumentation available for detection. The table below outlines characteristics of commonly used enhanced chemiluminescent (ECL) and SuperSignal chemiluminescent substrates.
Table 1. Recommended Pierce ECL and SuperSignal chemiluminescent substrates.
|Pierce ECL substrate||Pierce ECL Plus substrate||SuperSignal West Pico PLUS substrate||SuperSignal West Dura substrate||SuperSignal West Femto substrate|
|Detection level||Low- to midpicogram||Low-picogram||Low-picogram to high-femtogram||Mid-femtogram||Low- to midfemtogram|
|Signal duration||0.5–2 hr||5 hr||6–24 hr||24 hr||8 hr|
|Detection methods||X-ray film, CCD imager||X-ray film, CCD imager, fluorescence imager||X-ray film, CCD imager||X-ray film, CCD imager||X-ray film, CCD imager|
|Select when …||... target and sample are abundant||... target is less abundant, sample is limited, and you need chemifluorescent detection||... target is less abundant, sample is limited, and you need more sensitivity than an entry-level ECL substrate provides||... target is less abundant, sample is limited, and you are using CCD image capture||... target is least abundant, sample is precious, and you need maximum sensitivity|
|Value to you||Low cost; easy to switch from other entry-level ECL substrates||Best detection flexibility with chemifluorescent detection option||Best value;
works for majority of western blots
|Best signal duration||Best sensitivity|
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.
Figure 2. 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.
- Overview of Western Blotting
- Western Blotting Handbook
- Stripping and Reprobing Western Blots
- Protein Electrophoresis and Western Blotting Support Center
- Tech Tip #32: Guide to enzyme substrates for Western blotting
- Tech Tip: Chemiluminescent western blotting technical guide and protocols
- Tech Tip #24: Optimize antigen and antibody concentrations for Western blots
Technical aspects of chemiluminescent western blotting
Western blotting is a powerful, routine application; however, capturing a chemiluminescent signal can be challenging since western blotting comprises a series of steps that require specific skills to perform, failure to capture signal can be caused by many factors. With so many variables (Table 2), troubleshooting a problem blot can be likened to searching for a “needle in a haystack”. The traditional protocol is often ineffective in detecting a specific protein. Therefore, troubleshooting guides are frequently needed to help optimize results. For an overview of factors affecting western blot results review Table 2. To obtain western blot troubleshooting information visit, Western Blot Detection Support — Troubleshooting.
Table 2. Factors that affect western blotting results.
|Target antigen||Conformation, stability, available epitope(s)|
|Polyacrylamide gel||Manufacturer, percent polyacrylamide, age, lot|
|Membrane||Manufacturer, type, lot|
|Primary antibody||Specificity, titer, affinity, incubation time and temperature|
|HRP conjugate||Enzyme activation level and activity, source animal, concentration, detergent|
|Blocking buffer||Type, concentration, cross-reactivity|
|Washes||Buffer, volume, duration, frequency|
|Substrate||Sensitivity, manufacturer lot, age|
|Detection method||Film age, imaging instrument manufacturer, exposure time|
Signal intensity and duration
Under optimal western blotting conditions, a chemiluminescent signal can last for 6–24 hr. The level and duration of light generation depends on the specific substrate being used and the enzyme-to-substrate ratio in the system. Although the amount of substrate on a blot is relatively constant, the amount of enzyme present depends on the amount added and other factors (Table 2). Too much enzyme conjugate applied to a western blot system is one of the biggest causes of signal variability, dark background, short signal duration, and low sensitivity. A signal emission curve that decays slowly (Figure 3) is desirable as it demonstrates that each component of the system has been optimized and allows reproducible results. A signal that decays too quickly can cause variability, low sensitivity, high background, and problems with signal documentation. A long-lasting signal minimizes variability in results due to transfer efficiency, different manufacturer lots of substrate, and other factors. The oxidation reaction of the HRP molecules with luminol in the substrate produces free radicals in addition to the light being produced. An abundance of HRP in the system will create an abundance of free radicals, speeding the probability of HRP inactivation. Free radicals may also damage the antigen, antibodies, and the membrane, resulting in reduced effectiveness of re-probing. The figure below provides an example of signal emission curves generated with short- and long- duration substrates.
Figure 3. A comparison of signal emission curves for short- and longduration substrates. When there is an enzyme present in a western blot system, signal output peaks soon after substrate application and rapidly exhausts the substrate (Signal 1). In an optimal system, the signal emission peaks approximately 5 min after applying the substrate and plateaus for several hours (Signal 2).
Direct and indirect methods
Direct detection uses a labeled primary antibody. Because incubation with a secondary antibody is eliminated, this strategy takes less time than a classic western blot. Additionally, background signal from cross-reactivity with the secondary antibody is eliminated. Direct detection also enables probing for multiple targets simultaneously. Labeling a primary antibody, however, sometimes has an adverse effect on its immunoreactivity, and allows for no signal amplification. As the direct method is generally less sensitive than an indirect detection, it is most useful when the target is relatively abundant. One option is biotinylating the primary antibody, which is an indirect method that both amplifies the signal and eliminates the need for a secondary antibody. Labeling with biotinylation reagents typically results in more than one biotin moiety per antibody molecule. Each biotin moiety is capable of interacting with enzyme-conjugated avidin, streptavidin, or Thermo Scientific NeutrAvidin Protein (Cat. No. 31000). Essentially, the biotin-binding conjugate replaces a secondary antibody, and its appropriate molar concentration is the same as if a secondary antibody were used. The small size of biotinylation reagents (typically less than 2 kDa) compared to enzymes (40 kDa for HRP; 140 kDa for AP) often reduces steric hindrance, allowing greater functionality of the labeled primary antibody.
Figure 4. Difference between direct and indirect protein detection in an assay involving detection with specific antibodies (e.g., an immunoassay). The surface on which the target protein is bound and immobilized is either a membrane (western blot) or a microplate (antibody).
Occasionally, an antibody to a specific antigen is unavailable or unsuitable for western blot analysis. Target protein–specific detection by blotting is also possible if a corresponding binding partner is available for use as a probe. This type of application, referred to as a far-western blot, is routinely used for the discovery or confirmation of a protein–protein interaction. There are numerous variations in western blotting detection that include, but are not limited to, the previously mentioned strategies. In far-western blotting, labeled binding partners may be labeled by an in vitro translation reaction with 35S-labeled probe. Biotinylating the probe and detecting with a biotin-binding protein conjugate is also possible. This type of detection has the added advantage of signal amplification. However, when using this technique the probe should not be over-labeled, to prevent target detection from being compromised. Additionally, a recombinant probe can be expressed in bacteria with a tag such as GST, HA, c-Myc, or FLAG, in which detection occurs via a labeled antibody to the particular tag. As with other blotting applications, the far-western method is effective for both membrane and in-gel detection systems. The diagram below outlines with far-western workflow.
Figure 5. Diagram of far–western blot to analyze protein–protein interactions. In this example, a tagged bait protein is used to probe either the transfer membrane or a gel for the prey protein. Once bound, enzyme (horseradish peroxidase; HRP)-conjugated antibody that targets the bait tag is used to label the interaction, which is then detected by enzymatic chemiluminescence. This general approach can be adjusted, as shown in the table below, by using untagged bait protein that is detected by antibody, biotinylated bait protein that is detected by enzyme-conjugated streptavidin, or radiolabeled bait protein that is detected by exposure to film.
Chemiluminescence western blotting technical guide and protocols
This technical resource provides detailed descriptions about general principles involved in western blotting using chemiluminescent substrates. In addition to covering the use of relevant products in protein research. Importantly, the guide provides troubleshooting information and tips for optimizing chemiluminescent wester blotting procedures. The strategies and methods discussed are applicable to most enhanced chemiluminescence (ECL) western blot assays.
- Overview of Western Blotting
- Far Western Blot Analysis
- Overview of Protein Electrophoresis
- Blocking Buffers for Western Blotting and ELISA
- Enzyme Probes
- Overview of Detection Probes
- GST-tagged Proteins – Production and Purification
- Protein Research Handbook
- Protein Electrophoresis and Western Blotting Support Center
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.In addition to traditional data capturing methods, advanced digital imaging has become a powerful tool for measuring protein abundance and protein modifications. With the latest advances in imaging software and instrument sensitivity, quantitative western blot analysis is now easier to achieve.
View this video to learn more about western blot imaging technology
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.
Figure 6. 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.
- G.H. Thorpe, L.J. Kricka. (1986) Enhanced chemiluminescent reactions catalyzed by horseradish peroxidase. Methods in Enzymology 133:331–54.
- Alegria-Schaffer, A., et al. (2009) Performing and optimizing Western blots with an emphasis on chemiluminescent detection. Methods Enzymol 463:573–99.
- Gassmann, M., Granacher, B., Rohde, B. and Vogel, J. (2009) Quantifying Western blots: Pitfalls of densitometry. Electrophoresis 30:1845–55.
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