• My NEB
  • Print
  • PDF
  • BioLux® Gaussia Luciferase Flex Assay Kit

    Description

    The BioLux® Gaussia Luciferase Flex Assay Kit contains reagents necessary for assaying Gaussia Luciferase activity with adjustable light emission kinetics, most commonly secreted from mammalian cells transfected with GLuc expression plasmids.

    The BioLux GLuc Flex Assay Kit includes a higher concentration coelenterazine substrate and an additional stabilizer component, which allows the use of the assay in high throughput format or without the requirement of an injectorequipped luminometer. With the standard protocol 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 periods of time (Figure 2). 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 stability.

    Gaussia Luciferase is a luciferase reporter from the marine copepod Gaussia princeps (1,2). This luciferase, which does not require ATP, catalyzes the oxidation of the substrate coelenterazine in a reaction that produces light, and has considerable advantages over other luminescent reporter genes.

    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 can be used for the assay. Although most of the activity is secreted, the high sensitivity of GLuc allows measurements from the cellular fraction as well.

    Figure 1: The Photo-oxidation catalyzed by Gaussia Luciferase.
    Haddock, S.H.D., McDougall, C.M. and Case, J.F., The Bioluminescence Web Page, http://lifesci.ucsb.edu/~biolum/ (created 1997; updated 2005).
    Figure 2: GLuc kinetics with the BioLux GLuc Flex Assay Kit
    Reactions were setup without stabilizer and with the indicated amounts of stabilizer added. Time is shown in minutes after substrate addition in a total volume of 50 μl.
    Figure 3: Stability of Gaussia Luciferase at various temperatures.
    Growth media from GLuc-expressing cells (GLuc-sup) were incubated at 95°C and 55°C for 30 minutes and allowed to cool to room temperature (25°C) before assaying for GLuc activity.
    Figure 4: Stability of Gaussia Luciferase at 37°C over a period of 7 days.
    Growth media from GLuc-expressing cells grown in ±β-mercaptoethanol-containing media (GLuc-sup & GLucsup, β-mercaptoEtOH) were placed at 37°C and assayed everyday for a period of 7 days.
    Figure 5: Gaussia Luciferase assays.
    Figure 6: Gaussia activity after adding GLuc Flex assay solution to the sample.
    The GLuc Flex assay solution was prepared as suggested in the protocol. The measurements were taken at 1-second increments.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    BioLux® GLuc Flex Assay Buffer (1X)1X
    BioLux® GLuc Flex Stabilizer
    BioLux® GLuc Flex Substrate (100X)100X

    Advantages and Features

    Features

    • Gaussia Luciferase (GLuc) possesses a natural secretory signal and upon expression is secreted into the cell medium. Therefore, lysis of the cells is not necessary.
    • Gaussia Luciferase generates over 1000-fold higher bioluminescent signal intensity, than Firefly and Renilla Luciferases, making it an ideal transcriptional reporter (1). GLuc shows the highest reported activity of any characterized luciferases (2).
    • The secreted protein is thermally stable (Figure 3) and has extremely high activity in light production allowing for very sensitive assays (2).
    • The secreted GLuc is also very stable in the presence of 55 μM β-mercaptoethanol, which is typically used in culturing mouse stem cells (Figure 4).
    • The GLuc-containing samples (i.e. growth media or cell lysates after transfection) can be stored at -20°C for long-term storage or at 4°C for several days without loss of activity.
    • The stabilizer component of this assay system provides steady kinetics over a longer time period allowing users the time required for high-throughput analysis as well as manually delivered assays.

    Properties and Usage

    Storage Temperature

    -20°C

    Notes

    1. Because of the stability of GLuc, the activity measured in the growth media of a GLuc-expressing culture reflects the protein that has accumulated up to the time of sampling.
    2. The GLuc assay solution should be allowed to equilibrate at room temperature for 25 minutes (protect from light in a tightly capped tube/bottle) before adding to the sample.
    3. 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 the 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 compared to 30, 20 and 10 seconds of delay, the detection level is ~98% for 30 seconds of delay, ~93% for 20 seconds of delay & ~80% for 10 seconds of delay (Figure 6).
    4. Use the prepared assay solution within 24 hours. The unused portion of the assay solution should be tightly capped and stored at -20°C. It should be completely thawed in the dark at room temperature before use.
    5. 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 (growth media from untransfected cells) should be included to establish the background in the assay.
    6. 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 (e.g. 2 seconds instead of 5 seconds).
    7. When assaying the serial dilutions of a sample, it is best to assay the most diluted samples first & the most concentrated samples last. This will help to minimize false readings, i.e. cross talk effect in which signals from samples of high RLU cross into the next sample. The cross-talk effect seems to be more pronounced when white or black plates with clear-bottoms are used.
    8. BioLux GLuc Flex Assay Buffer and Stabilizer can be stored at 4°C.
    9. BioLux GLuc Flex Substrate must be tightly capped and stored at -20°C.

    References

    1. Verhaegen M. and Christopoulos T.K. (2002). Anal. Chem. 74, 4378-4385.
    2. Tannous, B.A., Kim, D.E., Fernandez, J.L., Weissleder, R., and Breakefield, X.O. (2005). Mol. Ther. 11, 435-443.
    3. Wu, C., Suzuki-Ogoh, C. and Ohmiya, Y. (2007). Bio Techniques. 42, 290-292.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Can the BioLux GLuc Flex Assay Kit be used without the addition of the Stabilizer?
    2. Can I use the BioLux GLuc assay systems (NEB #E3300, NEB #E3308) to assay Renilla luciferase activity in the cell lysates?
    3. Can I assay Gaussia and Firefly luciferase activities if reporter genes are co-transfected in the cells?
    4. Can I add GLuc assay working solution directly to the cells?
    5. Is the BioLux GLuc Flex Substrate stored at -20°C still good 3 months after the expiration date?
    6. Can I purchase the components of the assay kits individually?
    7. What is the concentration of coelenterazine in the assay kit?
    8. Can I buy the GLuc substrate at a higher concentration?
    9. Can I use more than 20µL of sample for assaying GLuc activity?
    10. Can I use a GLuc assay solution that has been sitting at room temperature for a few hours?
    11. Which BioLux GLuc assay kit (NEB #E3300, NEB #E3308) can I use for assaying GLuc activity in an in vivo model, i.e., injecting the assay solution into a mouse?
    12. After transfecting a GLuc-expressing vector into mammalian cells, how soon can the GLuc activity be detected?
    13. Once a GLuc-expressing vector is transiently transfected into mammalian cells, how long will the GLuc expression last?
    14. What is the half-life of the Gaussia luciferase (GLuc)?
    15. Is secreted GLuc active in a low pH environment and high temperatures?
    16. 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)?
    17. Can the BioLux GLuc assay systems (NEB #E3300 & NEB #E3308) be used to assay GLuc activity in GLuc-containing blood samples and GLuc samples containing high serum?
    1. Protocol I (Luminometers without injectors) (E3308)
    2. Protocol II (Injector-equipped luminometers) (E3308)

    Selection Tools

    Application Notes