Chemiluminescence Western Blotting Technical Guide and Protocols
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TECH TIP Chemiluminescent substrates Chemiluminescence western blotting technical guide and protocols Introduction Types and considerations of chemiluminescence Western blotting is a powerful and commonly used tool western blotting to identify and quantify a specific protein in a complex HRP is the most popular enzyme used in western mixture. As originally conceived by Towbin et al., the blotting and will be discussed throughout this document technique enables indirect detection of protein samples as our example. The most suitable western blotting immobilized on a nitrocellulose or polyvinylidene fluoride substrates for HRP are luminol-based, and they produce (PVDF) membrane. In a conventional western blot, a chemiluminescent signal. Chemiluminescence is a protein samples are first resolved by sodium dodecyl chemical reaction that produces energy released in the sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) form of light. In the presence of HRP and a peroxide buffer, and then electrophoretically transferred to the membrane. luminol oxidizes and forms an excited-state product that Following a blocking step, the membrane is probed emits light as it decays to the ground state. Light emission with a primary antibody (polyclonal or monoclonal) occurs only during the enzyme–substrate reaction and, that was raised against the antigen in question. After a therefore, once the substrate in proximity to the enzyme is subsequent washing step, the membrane is incubated with exhausted, signal output ceases. In contrast, colorimetric a secondary antibody conjugated to an enzyme that is substrates, such as diaminobenzidine (DAB), produce reactive toward the primary antibody. An enzyme such as precipitate that remains visible on the membrane even alkaline phosphatase (AP) or horseradish peroxidase (HRP) after the reaction has terminated. Several varieties of is necessary for signal generation. After a final wash step, Thermo Scientific™ Pierce™ ECL and SuperSignal™ the membrane is incubated with an appropriate enzyme chemiluminescent HRP substrates are available that substrate to generate a recordable signal. provide different levels of sensitivity for chemiluminescence western blotting. Refer to Table 1 to select the most This guide describes general principles and considerations appropriate HRP chemiluminescent substrate based involved in western blotting using chemiluminescent on the abundance of your target protein of interest, substrates. Relevant Thermo Scientific™ and Invitrogen™ abundance of sample containing the target protein, and products in protein research are referenced throughout, the level of sensitivity and type of instrumentation available but the strategies and methods discussed are applicable for detection. to most enhanced chemiluminescence (ECL) western blot assays. Table 1. Recommended Pierce ECL and SuperSignal chemiluminescent substrates. SuperSignal™ Pierce™ ECL Pierce ECL™ Plus West Pico PLUS SuperSignal™ West SuperSignal™ West substrate substrate substrate Dura substrate Femto substrate Detection Low- to mid- Low-picogram to Low- to mid- Low-picogram Mid-femtogram level picogram high-femtogram femtogram Signal 0.5–2 hr 5 hr 6–24 hr 24 hr 8 hr duration X-ray film, Detection X-ray film, X-ray film, X-ray film, X-ray film, CCD imager, methods CCD imager CCD imager CCD imager CCD imager fluorescence imager ... target is less ... target is less abundant, sample ... target is less ... target is least ... target and abundant, sample is is limited, and abundant, sample abundant, sample Select sample are limited, and you need you need more is limited, and you is precious, when … abundant chemifluorescent sensitivity than an are using CCD and you need detection entry-level ECL image capture maximum sensitivity substrate provides Low cost; easy to Best detection Best value; works switch from other flexibility with Value to you for majority of Best signal duration Best sensitivity entry-level ECL chemifluorescent western blots substrates detection option Don’t know where to start? Try our SuperSignal Signal intensity and duration West Pico Plus Chemiluminescent Substrate Under optimal western blotting conditions, a (Cat. No. 34580), designed to work for the majority of chemiluminescent signal can last for 6–24 hr. The level western blots. 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 Signal capture relatively constant, the amount of enzyme present depends Although western blotting is a powerful, routine application, on the amount added and other factors (Table 2). Too capturing that elusive chemiluminescent signal can be much enzyme conjugate applied to a western blot system frustrating. Since western blotting comprises a series is one of the greatest cause of signal variability, dark of steps that require specific skills to perform, failure to background, short signal duration, and low sensitivity. capture signal can be caused by many factors. With so many variables (Table 2), troubleshooting a problem blot A signal emission curve that decays slowly (Figure 1) is can be likened to searching for a “needle in a haystack”. desirable as it demonstrates that each component of The traditional protocol is often ineffective in detecting a the system has been optimized and allows reproducible specific protein. For example, the primary antibody may results. A signal that decays too quickly can cause not recognize the immobilized antigen in its denatured variability, low sensitivity, high background, and problems state. Although the protein can be kept in its native state by with signal documentation. A long-lasting signal minimizes using nondenaturing conditions, this makes determination variability in results due to transfer efficiency, different of the target molecular weight more challenging. Further, manufacturer lots of substrate, and other factors. exceptionally large or hydrophobic proteins are often difficult to transfer. Towbin’s original western blot protocol The oxidation reaction of the HRP molecules with the is therefore often modified to ensure target detection. Such luminol in the substrate produces free radicals in addition modifications may involve the use of different reagents for to the light being produced. An abundance of HRP in the indirect detection or a labeled primary probe for direct system will create an abundance of free radicals, speeding detection of the target. Sometimes transfer of protein from the probability of HRP inactivation. Free radicals may a gel to membrane is bypassed altogether and detection is also damage the antigen, antibodies, and the membrane, accomplished in the gel. resulting in reduced effectiveness of re-probing. Table 2. Factors that affect western blotting results. 120,000 Factor Variable characteristic 100,000 Target antigen Conformation, stability, available epitope(s) 80,000 Polyacrylamide Manufacturer, percent polyacrylamide, Signal 1 60,000 gel age, lot Signal 2 Membrane Manufacturer, type, lot 40,000 Signal intensity intensity Signal Primary antibody Specificity, titer, affinity, incubation time 20,000 and temperature 0 HRP conjugate Enzyme activation level and activity, source 1 2 3 4 5 6 7 animal, concentration, detergent Time (min) Blocking buffer Type, concentration, cross-reactivity Washes Buffer, volume, duration, frequency Figure 1. A comparison of signal emission curves for short- and long- duration substrates. When there is an enzyme present in a western blot Substrate Sensitivity, manufacturer lot, age system, signal output peaks soon after substrate application and rapidly Detection method Film age, imaging instrument manufacturer, exhausts the substrate (signal 1). In an optimal system, the signal emission exposure time peaks approximately 5 min after applying the substrate and plateaus for several hours (signal 2). 2 Direct and indirect methods with a biotin-binding protein conjugate is also possible. Direct detection uses a labeled primary antibody. Because This type of detection has the added advantage of signal incubation with a secondary antibody is eliminated, this amplification. However, when using this technique the strategy takes less time than a classic western blot. probe should not be over-labeled, to prevent target Additionally, background signal from cross-reactivity with detection from being compromised. Additionally, a the secondary antibody is eliminated. Direct detection recombinant probe can be expressed in bacteria with a also enables probing for multiple targets simultaneously. tag such as GST, HA, c-Myc, or FLAG, in which detection Labeling a primary antibody, however, sometimes has occurs via a labeled antibody to the particular tag. As an adverse effect on its immunoreactivity, and allows for with other blotting applications, the far-western method is no signal amplification. As the direct method is generally effective for both membrane and in-gel detection systems. less sensitive than an indirect detection, it is most useful when the target is relatively abundant. One option is Optimization biotinylating the primary antibody, which is an indirect Each western blot system must be optimized to obtain method that both amplifies the signal and eliminates consistently good results. Many factors influence signal the need for a secondary antibody. Labeling with intensity and duration, and each of these factors can be biotinylation reagents typically results in more than one optimized. Included below are discussions