![]() ![]() Blots where processed using the Bandmate Automated Western Blot Processor. Gels were transferred to nitrocellulose membranes using the iBlot 2 Transfer Device (P0, 7 min). A 3-fold serial dilution of A431 or A549 lysate was loaded onto a Bolt 4-12% Bis-Tris-Plus gel (NW04122BOX) and electrophoresed at 200V for 20 minutes. Obtain quantitative western blots with SuperSignal West Dura Substrate. Free radicals may also damage the antigen, antibodies, and the membrane, resulting in reduced effectiveness of re-probing. An abundance of HRP in the system will create an abundance of free radicals, speeding the probability of HRP inactivation. The oxidation reaction of the HRP molecules with the luminol in the substrate produces free radicals in addition to the light being produced. A long-lasting signal minimizes variability in results due to transfer efficiency, different manufacturer lots of substrate, and other factors. A signal that decays too quickly can cause variability, low sensitivity, high background, and problems with signal documentation. ![]() A signal emission curve that decays slowly is desirable as it demonstrates that each component of the system has been optimized and allows reproducible results. Too much enzyme conjugate applied to a western blot system is one of the greatest causes of signal variability, dark background, short signal duration, and low sensitivity. ![]() Although the amount of substrate on a blot is relatively constant, the amount of enzyme present depends on the amount of antibody-conjugate added. 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 Simply switch to Pierce ECL and save a bundle.ĮCL substrate – an enhanced chemiluminescent substrate for detection of horseradish peroxidase (HRP) activity from antibodies and other Western blot probesĪbout half the cost of other ECL Substrates – low overhead and a commitment to customer value enables Thermo Fisher Scientific to make this product available to you at a significantly lower price than other companies (these claims are based on the 2010 U.S.Detecting very low abundant protein targets, limited antibodies, or limited sample volumes Because the luminol and peroxide reagent-formulations are identical to other commercially available substrate products, one can switch to Pierce ECL without needing to optimize probing conditions or incubation protocols. Pierce ECL Western Blotting Substrate provides reliability and performance equivalent to other standard enhanced chemilumescent (ECL) substrates for detection of horseradish peroxidase (HRP) enzyme activity, yet costs much less. Thermo Scientific™ Pierce ECL Western Blotting Substrate is a value-priced, entry-level peroxidase substrate for enhanced chemiluminescence (ECL) that directly replaces costlier products without the need to re-optimize conditions. ![]()
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