![]() ![]() Carefully pick it up with flat forceps and dab it on a Kimwipe to remove excess buffer. Step 2 Incubated your blot with 2 Ab and washed it thoroughly with buffer. Each containing different enhancers to increase signal, linearity and duration compared to other western blot detection systems. Step 1 Wash your blot thoroughly with TBS-T, prepare the enhanced chemiluminescence (ECL) substrate according to the manufacturer’s instructions. 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. Compared to entry-substrates, SignalFire ECL Reagent boasts a more robust signal and extended duration of signal output. The two most common enzyme reporters that catalyze chemiluminescent reactions required for generating a recordable signal are horseradish peroxidase (HRP) and alkaline phosphatase (AP).ĮCL Substrates for High Sensitivity Western Blot DetectionĮnhanced chemiluminescence (ECL) substrates, are HRP substrates that are typically two-component systems consisting of a stable peroxide solution and an enhanced luminol solution. SignalFire ECL Reagent from Cell Signaling Technology (CST) is an enhanced chemiluminescent substrate capable of detecting picogram amounts of protein by western blot analysis. Recommended secondary antibody concentration: 1:50,000–1:250,000 dilution (4–20 ng/mL)Ĭhemiluminescent detection occurs when energy from a chemical reaction is released in the form of light.Recommended primary antibody concentration: 1:1,000–1:50,000 dilution (0.02–1.0 µg/mL) Thermo Scientific Pierce ECL Plus Substrate is an acridan-based chemiluminescent and chemifluorescent HRP substrate for western blot detection using X-ray.Compatibility: nitrocellulose and PVDF membranes.Stability: 24-hr working solution stability 1-year kit stability at room temperature.SuperSignal™ West Dura Features of Extended Duration Chemiluminescent Substrate ![]()
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