The level and duration of light generation depends on the specific substrate being used and the enzyme-to-substrate ratio in the system. Under optimal western blotting conditions, a chemiluminescent signal can last for 6–24 hr. Why do our ECL kits have recommended antibody dilutions? Use the table above to select the most appropriate ECL kit based on 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.ĭon’t know which ECL western blot kit is right for you? Use the chemiluminescent HRP substrate selection guide to choose the right HRP substrate for your research needs. Each containing different enhancers to increase signal, linearity and duration compared to other western blot detection systems. We offer six types of ECL substrates for western blot detection with horseradish peroxidase enzyme (HRP). Light emission occurs only during the enzyme-substrate reaction therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases. When incubated with a blot on which HRP-conjugated antibodies (or other probes) are bound, a chemical reaction emits light at 425 nm which can be captured with x-ray film, CCD camera imaging devices and phosphorimagers that detect chemiluminescence. To make a working solution, the two components are mixed together. Same signal and lower price than other standard ECL substratesĮnhanced chemiluminescence (ECL) substrates, are HRP substrates that are typically two-component systems consisting of a stable peroxide solution and an enhanced luminol solution. Good sensitivity with long duration and linearity for sensitive quantitationīetter signal and lower price than competing ECL substrates Highest sensitivity with less optimization required Performing quantitative western blots or long duration is neededįor routine western blots or new systems not yet optimizedĭetecting high abundant protein targets and sample is abundant Detecting very low abundant protein targets, limited antibodies, or limited sample volumes
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