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. 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. The primary antibody, which is specific to the target antigen, binds to the available epitope sites on the protein. Optimizing the antibody is a critical step to ensure proper signal to noise. Furthermore, individual lab protocols may direct researchers towards preferred, previously optimized conditions. 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. One of the most impactful technical factors in western blotting is optimizing the antibody dilutions. *Based on a 1mg/mL antibody concentration Most sensitivity with less optimization required Good sensitivity with optimized conditions Low cost easy to switch from other entry-level ECL substratesīest detection flexibility with chemifluorescent detection optionīest value works for majority of western blots target is least abundant, sample is precision, and you need maximum sensitivity target is least abundant, sample is precious, and you need maximum sensitivity target is less abundant, sample is limited, and you are using CCD image capture 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 need chemifluorescent detection X-ray film, CCD imager, fluorescence imager 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. 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. In most cases, to make a working solution, equal volumes of the two components are mixed together. The table also outlines characteristics of commonly used enhanced chemiluminescent (ECL) and SuperSignal chemiluminescent substrates.Ĭhemiluminescent substrates for HRP are most commonly two-component systems consisting of a stable peroxide solution and an enhanced substrate solution, often luminol-based. 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. Several varieties of Thermo Scientific Pierce ECL and SuperSignal chemiluminescent HRP substrates are available that provide different levels of sensitivity for chemiluminescence western blotting. Because its reaction rate remains linear, detection sensitivity can be improved by simply allowing a reaction to proceed for a longer time period.Īs HRP is the most popular enzyme used in western blotting it will be discussed throughout this article as our example. As a label for Western blotting, AP offers a distinct advantage over other enzymes. 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. 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. 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. In addition, its high turnover rate, good stability, low cost, and wide availability of substrates makes HRP the enzyme of choice for most applications. Antibody-HRP conjugates are superior to antibody-AP conjugates with respect to the specific activities of both the enzyme and antibody. HRP functions optimally at a near-neutral pH and can be inhibited by cyanides, sulfides and azides. 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.
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