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Gaussia Luciferase Assay reagent (GAR-2B)Stable version
*Call for special pricing on bulk purchases Description: The GAR-2BGaussiaLuciferase Assay Kit contains the reagents necessary for assayingGaussiaLuciferase (GLuc) activity, most commonly from cell culture supernatants.Gaussia Luciferase is a reporter luciferase from the marine copepodGaussia princeps (1,2).Gaussia Luciferase can be expressed in mammalian cells using reporter plasmids available from NEB (Refer to the Companion Products). This luciferase, which does not require ATP, catalyzes the oxidation of the substrate coelenterazine in a reaction that produces light (Figure 1), and has several advantages over other luminescent reporter genes. This kit includes an additional stabilizer component, which allows the use of the assay in high throughput format or without the requirement of an injector-equipped luminometer.This three-component assay system provides the user with 2 options: (a) use the assay without stabilizer for enhanced light output or (b) use with the desired amount of stabilizer for enhanced signal stability. The stabilizer component allows the use of the assay in high throughput format or without the requirement of an injector-luminometer. For standard assays giving the highest activity, the kit can be used with the GLuc substrate mixed in the assay buffer. With the stabilized assay protocols, the light emission decays slowly with a half-life of approximately 25 minutes. The addition of stabilizer decreases the absolute value of light output but confers signal stability over time (Fig 2). The luminescence measured from the supernatant of cultured cells transfected with a plasmid expressing GLuc is proportional to the amount of enzyme produced, which in turn, reflects the level of transcription. Alternatively, a cell lysate sample can be used for the assay. Although most of the GLuc is secreted, the high sensitivity of GLuc allows measurements from the cellular fraction. Figure 1: Photo-oxidation of coelenterazine catalyzed by Gaussia luciferase  Figure 2: GLuc kinetics using the GAR-2B Assay Kit in either standard or stabilized assay (ASSAY OF NATIVE GAUSSIA LUCIFERASE). FOR ASSAYING THE MORE STABLE GAUSSIA LUCIFERASE MUTANTS PLEASE USE THE GAR_2B REAGENT WITHOUT STABILIZER 
Assays were setup using assay solution without stabilizer or with the indicated amounts of stabilizer (5µL, 8µL or 10µL of stabilizer per 50µL GLuc assay solution). Figure 3: Gaussia Luciferase activity after adding GLuc assay solution containing stabilizer to a sample.
The following reagents are supplied with this product:
Advantages and Features
Notes: Because of the stability of GLuc, the activity measured in the growth media of GLuc-expressing culture reflects the protein that has accumulated up to the time of sampling. For the standard assay solution, i.e. solution that does not contain stabilizer, equilibration of the assay solution is not necessary.After adding the GLuc assay solution to the sample, we recommend a delay of 1-5 seconds before taking a measurement. Keeping the delay time consistent across experiments will ensure reproducibility. For the stabilized assay solution, i.e., the stabilizer-containing GLuc assay solution, the solution should be equilibrated at room temperature for 25 minutes (protect from light in a tightly capped tube/bottle) before adding to the sample. After adding the equilibrated GLuc assay solution to the sample, we recommend a delay time of 35-40 seconds before taking a measurement in order to reach maximum level of detection. This is especially important when the GLuc activity level is low (e.g. < e4 RLU). For example, the readout obtained after 35-40 seconds of delay is ~e4; when compare to 30, 20 and 10 seconds of delay, the readouts are as follows: ~2% decrease (for 30 seconds of delay), ~7% decrease (for 20 seconds of delay), & ~20% decrease (for 10 seconds of delay) in RLU (refer to Figure 5). Use the prepared GLuc assay solution within 2 hours. The linear range of the luminometer used for the assay must be established. This is easily done by assaying serial dilutions of a sample. In addition, the assay solution itself, as well as the conditioned media (i.e. growth media from untransfected cells) should be included in the assay to establish the background signal in the assay. If excess activity for the instrument range is found, the sample should be diluted in either PBS or 10% serum-containing media. The integration time can also be reduced. When assaying the serial dilution of a sample, it is best to assay the most diluted samples first and the most concentrated samples last. This will help to minimize false readings, i.e., cross-talk effect (signals from samples of high RLU cross into that of the next sample). The cross-talk effect seems to be more pronounced when plates (white or black) with clear-bottoms are used. The Gaussia Luciferase Assay Buffer and the Gaussia luciferase Stabilizer can be stored at 4˚C while the GLuc Substrate must be stored at -20˚C. References:
Assay Protocol: Standard Assay Protocol I (Luminometers without injectors)
Standard Assay Protocol II (Luminometers with injectors))
Stabilized Assay Protocol I (Luminometers without injectors)
Stabilized Assay Protocol II (Injector-equipped luminometers)
* Approximately 90% of GLuc is secreted out into the growth media after transfection and thus, the GLuc activity is typically assayed from the supernatant (i.e. growth media of GLuc-transfected cells). However, as long as the cells are alive, approximately 10% of GLuc is present inside the cells. Therefore, GLuc activity can also be assayed from the cell lysate. We recommend that the cell lysates be prepared by using Luciferase Cell Lysis Buffer (5X CLR-01), since this lysis buffer is designed to be compatible with Gaussia, Cypridina and Renilla, Firefly luciferase Measurement of intracellular Gaussia luciferase activity: Protocol to measure intracellular luciferase activit:
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