Thermo Scientific SuperSignal ELISA Pico Chemiluminescent Substrate is optimized to generate an intense light signal and provide exceptional performance in luminometer-based assays.
SuperSignal ELISA Pico Chemiluminescent Substrate is optimized for luminometer-based assays to generate an intense light signal. Researchers looking for greater sensitivity in their ELISAs or any other solution-based assay are now able to use chemiluminescent substrates for enzyme detection and quantification. These ELISAs can take place in either a test tube or a microplate and are quantified by measuring relative light units (RLU) in a luminometer. SuperSignal ELISA Pico Chemiluminescent Substrate was developed for researchers who need high sensitivity at an economical price.
- Immediate light generation – intense signal is produced immediately at room temperature or at 37°C; emits light at 425nm
- High signal:noise ratio – minimal background
- Low picogram sensitivity – detect proteins in your ELISAs down to the picogram levels
- Room temperature storage – a consistent product with ambient shipping and no need to store at 4°C
- 8-hour working solution stability – consistent performance of the working solution over an 8-hour period with only a 10% decrease in activity at 24 hours
- Flexible – signal can be read in black or white opaque plates
When energy in the form of light is released from a substance because of a chemical reaction, the process is called chemiluminescence. Luminol is one of the most widely used chemiluminescent reagents and its oxidation by peroxide results in creation of an excited state product called 3-aminophthalate. This product decays to a lower energy state by releasing photons of light.
|The chemiluminescent luminol reaction used by the Thermo Scientific SuperSignal ELISA Pico Substrate for horseradish peroxidase.
Chemiluminescent substrates differ from other substrates in that the light detected is a transient product of the reaction that is only present while the enzyme-substrate reaction is occurring. This is in contrast to substrates that produce a stable, colored product; these colors persist in the well after the enzyme-substrate reaction has terminated.
In a chemiluminescent ELISA, the substrate is the limiting reagent in the reaction; as it is exhausted, light production decreases and eventually ceases. A well optimized procedure using the proper antibody dilutions will produce a stable output of light, allowing consistent and sensitive detection of proteins. When the antibody is not diluted sufficiently, too much enzyme is present and the substrate is used up quickly. A stable output of light will never be achieved. This is the single greatest cause of variability in chemiluminescent ELISAs. To avoid this problem, it is crucial to optimize the amount of antibody used for detection. Antibody suppliers typically suggest a dilution range for using their antibody in an ELISA. This dilution range is often appropriate for experiments detected with a relatively insensitive chromogenic substrate, but a much greater dilution is generally required for optimum performance with a sensitive chemiluminescent substrate.
|Immediate light generation with Thermo Scientific SuperSignal ELISA Pico Chemiluminescent Substrate. 200pg of biotinylated HRP or biotinylated AP were added to separate wells of Thermo Scientific Pierce NeutrAvidin Coated White Polystyrene Plates. The plates were then incubated for 30 minutes at room temperature (RT) on a plate shaker and then each well was washed three times with BupH Tris Buffered Saline. Working solutions of chemiluminescent substrates were prepared according to the manufacturers’ instructions. For SuperSignal ELISA Pico Chemiluminescent Substrate and another luminol-based system (Brand A), 100µL of each substrate working solution was added to the appropriate plate well. For the dioxetane-based system, wells were washed with 1X Assay Buffer. Next, 100µL of the dioxetane working solution was added to the appropriate plate well. All plates were incubated on a plate shaker at RT for 1 minute. Plates were then read on a Dynex MLX Microtiter Plate Luminometer with a 0.2 second read time per well. Several readings were taken over a 30-minute period.
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