Chemiluminescent detection for western blotting is popular because it offers several advantages over other detection methods. Using chemiluminescence allows multiple 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 potential sensitivity of any available detection method for western blotting. These advantages have allowed chemiluminescence to become the detection method of choice in most protein laboratories.
Western blotting is a powerful technique that enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membranes. 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 that was raised against the antigen in question. After a subsequent washing step, the membrane is incubated with a conjugated secondary antibody that is reactive toward the primary antibody. The two most common enzymes conjugated to secondary antibodies are horseradish peroxidase (HRP) and alkaline phosphatase (AP). These enzymes catalyze the chemical reaction for generating a recordable signal in the form of light.
Chemiluminescent substrates for HRP and AP differ from other substrates in that the light detected is a transient product of the reaction that is only present while the enzyme-substrate reaction is occurring. This contrasts with chromogenic substrates that produce a stable, colored product; these colored precipitates remain on the membrane after the enzyme-substrate reaction has terminated. On a chemiluminescent western blot, the substrate is the limiting reagent in the reaction; as the substrate is exhausted, light production decreases and eventually ceases. A well-optimized procedure using proper antibody dilutions will produce a stable output of light for several hours, allowing consistent and sensitive detection of proteins.
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.
Chemiluminescent reaction of HRP substrate- 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.
Chemiluminescent reaction of AP substrate- CDP-Star. CDP-Star is dephosphorylated by AP to yield meta-stable dioxetane phenolate anion intermediate that decomposes and emits light with a maximum intensity at a wavelength of 475 nm. Light emission occurs only during the enzyme-substrate reaction.
|HRP substrates (Horseradish peroxidase)||AP substrates (Alkaline phosphatase)|
|Signal generation||Immediate||Gradually increases with signal maximum at ~30-60 minutes|
|Signal duration||Up to 24 h||24-96 hours|
|Considerations||Compatible with common buffers such as TBS and PBS||Not compatible with phosphate buffers|
|When to use||Antibodies or Probes conjugated to HRP||Antibodies or Probes conjugated to AP|
Horseradish peroxidase (HRP) catalyzes the oxidation of substrates by hydrogen peroxide, resulting in a colored or fluorescent product and the release of light as a by-product of the reaction. HRP functions optimally at a near-neutral pH and can be inhibited by cyanides, sulfides and azides. Antibody-HRP conjugates are superior to antibody-AP conjugates with respect to the specific activities of both the enzyme and antibody. In addition, its high turnover rate, good stability, low cost, and wide availability of substrates makes HRP the enzyme of choice for most applications. Because of the relatively small size of the HRP enzyme, further increases in sensitivity may be achieved by using poly-HRP conjugated secondary antibodies and may eliminate the need for using ABC-type amplification systems for some researchers. Alkaline phosphatase (AP) catalyzes the hydrolysis of phosphate groups from a substrate molecule resulting in a colored or fluorescent product and the release of light as a byproduct of the reaction. AP has optimal enzymatic activity at a basic pH (pH 8–10) and can be inhibited by cyanides, arsenate, inorganic phosphate and divalent cation chelators, such as EDTA. As a label for Western blotting, AP offers a distinct advantage over other enzymes. Because its reaction rate remains linear, detection sensitivity can be improved by simply allowing a reaction to proceed for a longer time period.
As HRP is the most popular enzyme used in western blotting it will be discussed throughout this article 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 also outlines characteristics of commonly used enhanced chemiluminescent (ECL) and SuperSignal chemiluminescent substrates.
Chemiluminescent substrates for HRP are most commonly two-component systems consisting of a stable peroxide solution and an enhanced substrate solution, often luminol-based. In most cases, to make a working solution, equal volumes of the two components are mixed together. When incubated with a blot on which HRP-conjugated antibodies (or other probes) are bound, a chemical reaction emits light at 425nm which can be captured with X-ray film and CCD camera imaging devices that detect chemiluminescence. Although X-ray film provides qualitative and semi-quantitative data and is useful to confirm the presence of target proteins, cooled CCD camera based imagers offer the advantages of quantitative analysis, rapid data capture, higher sensitivity, greater resolution and a larger dynamic range than film.
Table 1. Thermo Scientific chemiluminescent HRP substrates.
|Pierce ECL substrate||Pierce ECL Plus substrate||SuperSignal West Pico PLUS substrate||SuperSignal West Dura substrate||SuperSignal West Femto substrate||SuperSignal West Atto substrate|
|Detection level||Low- to midpicogram||Low-picogram||Low-picogram to high-femtogram||Mid-femtogram||Low- to midfemtogram||Low femtogram- high attogram|
|Signal duration||0.5–2 hr||5 hr||6–24 hr||24 hr||8 hr||6 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||X-ray film, CCD imager|
|Recommended primary antibody dilution*||1:1,000||1:1,000||1:1,000||1:5,000||1:5,000||1:5,000|
|Recommended secondary antibody dilution*||1:25,000 - 1:200,000||1:1,000 - 1:15,000||1:20,000 - 1:100,000||1:50,000 - 1:250,000||1:100,000 - 1:500,000||1:100,000 - 1:250,000|
|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||... target is least abundant, sample is precision, 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||Good sensitivity with optimized conditions||Most sensitivity with less optimization required|
|*Based on a 1mg/mL antibody concentration|
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. 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, SuperSignal West Pico PLUS Chemiluminescent Substrate enables picogram- to high femtogram–level protein detection by western blot analysis.
One of the most impactful technical factors in western blotting is optimizing the antibody dilutions. Many factors may influence the desired antibody dilution including volume of antibody available, antibody specificity for the antigen, protein abundance and choice of available substrates. Furthermore, individual lab protocols may direct researchers towards preferred, previously optimized conditions. Optimizing the antibody is a critical step to ensure proper signal to noise. The primary antibody, which is specific to the target antigen, binds to the available epitope sites on the protein. After a series of washing steps which helps remove unbound primary antibody, a secondary antibody, conjugated to an enzyme such as HRP, is incubated and binds to the primary antibody. Multiple secondary antibodies can bind to a single primary antibody- resulting in an amplification process whereby more light-producing labels can react and produce higher detectable signal. Too much primary or secondary antibody can cause oversaturation of the blot or excessive background noise. Also, too much antibody can lead to rapid signal formation followed by degradation – its possible for the signal to fade quickly before one could capture the signal on film or with an imaging system. These problems make it extremely difficult to properly analyze and make conclusions from the experiment. The table below can help provide general guidance on antibody dilutions depending on the protein target or sample abundance, antibody conditions, and substrate of choice. It is also important to note that when switching from one substrate to another, one may need to reoptimize their antibody dilutions based on the vendor’s recommendations.
Antibody dilution recommendations
|Sample||Antigen||Antibody||Recommended substrate||Primary||Secondary||Special notes|
|Sample is abundant||Antigen is abundant||Antibody quality is good||SuperSignal West Pico PLUS||1:1K||1:100K|
|Sample is abundant||Antigen is abundant is unknown||Antibody quality is unknown||SuperSignal West Pico PLUS||1:1K||1:100K||If no bands are seen, the blot can be washed and re-imaged with Atto|
|Sample is abundant||Antigen abundance is low||Antibody quality is poor||SuperSignal West Atto substrate||1:1K||1:100K|
|Sample is abundant||Antigen abundance is low||Antibody quality is good||SuperSignal West Atto substrate||1:2K||1:250K||Assumes normal primary usage is 1:1K|
|Sample is abundant||Antigen abundance is low||Antibody is limited||SuperSignal West Atto substrate||1:5K||1:100K||Assumes normal primary usage is 1:1K|
|Sample is abundant||Antigen is abundant||Antibody is limited||SuperSignal West Atto substrate||1:20K||1:250K||Assumes normal primary usage is 1:1K|
|Sample is limited||Antigen is abundant||Antibody quality is good||SuperSignal West Pico PLUS||1:1K||1:100K||Alternatively, could load less and use Atto at same antibody concentrations|
|Sample is limited||Antigen abundance is unknown||Antibody quality is unknown||SuperSignal West Pico PLUS||1:1K||1:100K||If no bands are seen, the blot can be washed and re-imaged with Atto|
|Sample is limited||Antigen abundance is low||Antibody quality is good||SuperSignal West Atto substrate||1:1K||1:250K|
|Sample is limited||Antigen abundance is low||Antibody quality is poor||SuperSignal West Atto substrate||1:1K||1:100K|
|*Based on a 1mg/mL antibody concentration|
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 — Troubleshooting.
Table 2. Factors that affect western blotting results.
|Factor||Variable characteristic||Quick Tips|
|Target antigen||Conformation, stability, available epitope(s)|
|Polyacrylamide gel||Manufacturer, percent polyacrylamide, age, lot||Use Bis-Tris gels for low/medium abundant proteins, use Tris-glycine gels for high abundant proteins, Tricine gels for low molecular weight proteins, and Tris-Acetate gels for high molecular weight proteins|
|Membrane||Manufacturer, type, lot|
|Primary antibody||Specificity, titer, affinity, incubation time and temperature||Use antibodies validated specially for western blotting|
|HRP conjugate||Enzyme activation level and activity, source animal, concentration, detergent|
|Blocking buffer||Type, concentration, cross-reactivity|
|Washes||Buffer, volume, duration, frequency||Use minimum of 6x 5 minutes washes prior to substrate addition to thoroughly remove unbound secondary antibody|
|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 (see figure below) 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.
A comparison of signal emission curves for short- and long duration 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.
Direct and indirect protein detection and signal amplification by avidin-biotin complex formation in an assay involving detection with specific antibodies (e.g., an immunoassay).
Occasionally, an antibody to a specific antigen is unavailable or unsuitable for western blot analysis. Target protein–specific detection by blotting is 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.
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.
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 capture and analysis, higher sensitivity, greater resolution and a larger dynamic range than film. Additionally, there is no need for dedicated darkroom space and developer equipment. Imaging instruments can be placed directly on a bench alongside other lab equipment.
CCD cameras for chemiluminescent image capture
|Dynamic range with >4 orders of magnitude||Extended exposures (>60 minutes) can result in increased background from camera noise|
|Smart algorithms help determine optimal exposure time|
|Streamlined image analysis and storage|
|Instant visualization of results|
|iBright Imaging systems feature a 9.1 MP cooled CCD camera that provides high sensitivity and dynamic range to help enable the detection of subtle differences in samples.|
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. Furthermore, judging the optimal exposure time is highly subjective, as its difficult to visually determine the precise point at which the best balance of signal to noise is achieved. Repeated bouts of exposing the film and developing the film are often performed, which can add up to a considerable investment of time. After the data is captured on film, the film is often scanned to make a digital copy of the data, which is then exported to analysis software to facilitate further analysis, such as densitometry and molecular weight estimation.
One of the biggest benefits of digital imaging systems is the ability to fine-tune the exposure time (some systems offer the ability to adjust exposure time to the thousandth of a second). In addition, many systems offer specific auto-exposure setting which employ algorithms that automatically determine the optimal exposure time, which not only captures an image with optimal signal-to-noise, but also saves a considerable amount of time versus the multiple exposures often required with film. Since the data is captured digitally, it can be easily exported to analysis software (eliminating the scanning step with film). Some of the most modern systems offer the ability to perform analysis directly on the instrument. These conveniences are making digital imaging a popular replacement for film-based data capture.
Molecular weight markers
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, 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).
iBright Prestained Protein Ladder contains twelve recombinant proteins, ten (11 to 250 kDa) that are blue-stained and fluor-labeled for direct and near-IR fluorescent visualization and protein sizing, and two (30 kDa and 80 kDa) that are unstained and contain IgG binding sites to bind the primary and secondary antibodies used for chemiluminescent and fluorescent detection of the target protein.
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.
Explore: Western blot protein ladders
- 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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