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  • BioLux® Gaussia Luciferase Assay Kit

    Description

    The 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 emits light (Figure 1), and has considerable advantages over other reporter genes.

    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: 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 3: 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 4: Gaussia & Renilla assay systems.
    Light reaction of GLuc secreted from mammalian cells using the Gaussia Luciferase Assay Kit versus a commercially available Renilla Assay Kit.
    Figure 5: Gaussia Luciferase assays.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    BioLux® GLuc Assay Buffer1X
    BioLux® GLuc Substrate100X

    Advantages and Features

    Features

    • Gaussia Luciferase possesses a natural secretory signal and upon expression is secreted into the cell medium. Therefore, lysis of the cells is not necessary.
    • GLuc generates over 1000-fold higher bioluminescent signal intensity, when compared to Firefly and Renilla Luciferases, making it an ideal transcriptional reporter (2).
    • The secreted protein is thermally stable (Figure 2) and has extremely high activity in light production allowing for very sensitive assays.
    • The secreted GLuc is also very stable in the presence of 55µM ß-mercaptoethanol, which is typically used in culturing mouse stem cells (Figure 3).
    • The GLuc-containing samples (i.e. growth media or cell lysates) can be stored at -20˚C for long-term storage or at 4˚C for several days without loss of activity.
    • The Gaussia Luciferase Assay Kit has been designed to stabilize the light production with robust kinetics, which gives more consistent results (Figure 4).
    • A simple three-step process (Figure 5) makes it easy to assay multiple samples collected data at different time points.



    Properties and Usage

    Storage Temperature

    -20°C

    Notes

    1. 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.
    2. Equilibration of the GLuc assay solution is not necessary.
    3. After adding the GLuc assay solution to the sample, we recommend a delay time of 5–15 seconds before taking a measurement. Keeping the delay time consistent across experiments will ensure reproducibility.
    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) to 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 (i.e. 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 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 Gaussia Luciferase Assay Buffer can be stored at 4°C.
    9. BioLux Gaussia Luciferase 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). BioTechniques. 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 I use the BioLux GLuc assay systems (NEB #E3300, NEB #E3308) to assay Renilla luciferase activity in the cell lysates?
    2. Can I assay Gaussia and Renilla luciferase activities if reporter genes are co-transfected in the cells?
    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 Gaussia Luciferase 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) (E3300)
    2. Protocol II (Injector-equipped luminometers) (E3300)

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    Application Notes