DescriptionThe BioLux® Gaussia Luciferase Assay Kit contains the reagents necessary for assaying Gaussia Luciferase (GLuc) activity, most commonly from cell culture supernatants. Gaussia Luciferase is a reporter luciferase from the marine copepod Gaussia 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 considerable advantages over other luminescent reporter genes.
This kit now 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 (Figure 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.
- Gaussia Luciferase (GLuc) possesses a natural secretory signal and upon expression is secreted into the cell medium. Therefore, lysing cells in order to assay GLuc activity is not necessary. As a result, GLuc is an ideal reporter gene for time course studies (3).
- GLuc generates over 1000-fold higher bioluminescent signal intensity, when compared to Firefly and Renilla luciferases, making it an ideal transcriptional reporter (3).
- GLuc show the highest reported activity of any characterized luciferases (4).
- The secreted protein is thermally stable (Figure 3) and has extremely high activity in light production for very sensitive assays (2).
- The secreted GLuc is also very stable in the presence of 55µM ß-mercaptoethanol, which is typically used in culturing mouse stem cells (Figure 4).
- The GLuc-containing samples, i.e., growth media or cell lysates, can be stored at -20˚C for long-term storage or 4˚C for several days without loss of activity.
- The stabilizer component of the this assay system provides steady kinetics over a long period of time allowing users the time required for high throughput analysis as well as manually delivered assays.
- BioLux® GLuc Assay Buffer
- BioLux® GLuc Substrate
- BioLux® GLuc Flex Stabilizer
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|BioLux® GLuc Assay Buffer||4||1X|
|BioLux® GLuc Substrate||-20||100X|
|BioLux® GLuc Stabilizer||4||100X|
Properties and Usage
- 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 24 hours. The unused portion of the assay solution should tightly capped and stored at -20˚C. It should be completely thawed (in the dark) at room temperature before use.
- 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 BioLux GLuc Assay Buffer and the BioLux GLuc Stabilizer can be stored at 4˚C while the BioLux GLuc Substrate must be stored at -20˚C.
- Verhaegen M. and Christopoulos T.K. (2002). Anal. Chem. 74, 4378-4385.
- Tannous, B.A., Kim, D.E., Fernandez, J.L., Weissleder, R., and Breakeﬁeld, X.O. (2005). Mol. Ther. 11, 435-443.
- Wu, C., Suzuki-Ogoh, C. and Ohmiya, Y. (2007). BioTechniques. 42, 290-292.
- Goerke, A., Loening, A., et al. (2008). Metabolic Engineering. 10, 187–200.
- Can I use the BioLux GLuc assay systems (NEB #E3300, NEB #E3308) to assay Renilla luciferase activity in the cell lysates?
- Can I assay Gaussia and Renilla luciferase activities if reporter genes are co-transfected in the cells?
- Can I assay Gaussia and Firefly luciferase activities if reporter genes are co-transfected in the cells?
- Can I add GLuc assay working solution directly to the cells?
- Is the BioLux Gaussia Luciferase Substrate stored at -20°C still good 3 months after the expiration date?
- Can I purchase the components of the assay kits individually?
- What is the concentration of coelenterazine in the assay kit?
- Can I buy the GLuc substrate at a higher concentration?
- Can I use more than 20µL of sample for assaying GLuc activity?
- Can I use a GLuc assay solution that has been sitting at room temperature for a few hours?
- Can I use the BioLux Gaussia Luciferase Assay Kit to assay GLuc activity in an in vivo model, i.e., injecting the assay solution into a mouse?
- After transfecting a GLuc-expressing vector into mammalian cells, how soon can the GLuc activity be detected?
- Once a GLuc-expressing vector is transiently transfected into mammalian cells, how long will the GLuc expression last?
- What is the half-life of the Gaussia luciferase (GLuc)?
- Is secreted GLuc active in a low pH environment and high temperatures?
- I used a lysis buffer from another company to make cell lysates so can you tell me if this lysis buffer is compatible with the BioLux GLuc assay systems (NEB #E3300 & NEB #E3308)?
- Can the BioLux GLuc Assay Kit (E3300) be used to assay GLuc activity in GLuc-containing blood samples and GLuc samples containing high serum?
- Can the BioLux GLuc Assay Kit be used for measuring Renilla luciferase activity?
- Where can I find the NEB’s kit cited as BioLux GLuc Flex Assay Kit, E3308?