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  • BioLux® Cypridina Luciferase Assay Kit

    Description

    The BioLux Cypridina Luciferase Assay Kit contains the reagents necessary for assaying Cypridina Luciferase activity. Cypridina is a new secreted luciferase reporter from the ostracod Cypridina noctiluca. This luciferase does not require ATP and catalyzes the oxidation of its luciferin substrate in a photochemical reaction (1,2) (Figure 1). The substrate for Cypridina is different than coelenterazine, which is the common substrate of other marine luciferases including Renilla and Gaussia.

    Properties of Cypridina Luciferase: 

    Cypridina Luciferase is secreted from cells by virtue of its natural signal peptide and its luminescence can be measured from the supernatant of transfected cells. Therefore, cell lysis is not necessary (Figure 2). 

    Secreted CLuc is a very stable protein. Because of this property, the activity measured from the supernatant reflects the amount of protein accumulated up to the time of sampling. Multiple samples can therefore be obtained from the same transfected cells (Figure 2). 

    The CLuc assay is very sensitive, allowing detection of very small amounts of Cypridina Luciferase activity (Figure 3). 

    Secreted CLuc is thermally stable at 55°C (Figure 4A), which is the typical inactivation temperature of most viruses. Secreted CLuc remains active in the presence of β-mercaptoethanol, which is commonly present in the complete culture medium of mouse ES cells (Figure 4B). 

    Although the light reaction catalyzed by Cypridina has an initial emission half-life of approximately 5 minutes, light production continues to decay slowly, and is readily detectable 25 minutes after substrate addition (Figure 5).

    Kit Includes: #E3309S #E3309L
    BioLux Cypridina Luciferase Assay Buffer  5 ml 2 x 25 ml
    BioLux Cypridina Luciferase Substrate Solvent* 0.5 ml .0.5 ml
    BioLux Cypridina Luciferase Substrate lyophilized powder
    (Kit components are not sold separately)

    *
    The 0.5 ml BioLux Cypridina Luciferase Substrate Solvent must be completely thawed and diluted with absolute ethanol (not included) before using it to make the 100X substrate solution (please refer to the section of Reconstitution of BioLux Cypridina Luciferase Substrate).

    Figure 1: The photochemical reaction catalyzed by Cypridina Luciferase.
    Figure 2: Activity of secreted CLuc at different time points.
    Supernatants of transfected cells were collected and medium was replaced each day for 4 days. These supernatants were stored at -20°C until the last samples were obtained. Relative Light Units, RLU.
    Figure 3: Sensitivity of Cypridina Luciferase assay.
    The CLuc activity was assayed from 10-fold serial dilutions of the supernatant from a stable CLuc-expressing cell line.
    Figure 4: Stability of Cypridina Luciferase.
    (A) Supernatant obtained from stable CLuc-expressing cells was incubated at 55°C or 95°C for 30 minutes and allowed to cool to room temperature (25°C). Five-fold serial dilutions of supernatant were assayed for CLuc activity. The control is the supernatant of the parental cells from which the stable cell line was created. (B) Supernatants from stable CLuc-expressing cells, grown in medium with or without β-mercaptoethanol, were incubated at 37°C and assayed each day for 7 days.
    Figure 5: Kinetics of light emission.
    The Cypridina Luciferase kinetic assay was performed with the supernatant from HeLa cells transiently
    transfected with a CLuc-expressing vector. The assay solution was prepared and incubated at room
    temperature for 30 minutes before use.
    Figure 6: Cypridina Luciferase Assays
    Figure 7: Activity of Cypridina Luciferase in the supernatant and lysate from a stable CLuc-expressing cell line.
    The CLuc activity was measured from 20 μl of supernatant (from 500 μl total volume) and from 20 μl of cell lysate (100 μl total lysate volume) of a stable CLuc-expressing cell line.

    Properties and Usage

    Storage Temperature

    -20°C

    Notes

    1. Store the BioLux Cypridina Luciferase Assay Buffer (1X) at -20°C for up to 2 years.
    2. The BioLux Cypridina Luciferase Assay Buffer (1X), the reconstituted CLuc substrate (100X solution) and the CLuc assay solution must be protected from light.
    3. The CLuc assay solution should be freshly prepared and incubated at room temperature for 30 minutes (protect from light) before use.
    4. The linear range of the luminometer must be established. This can be easily done by assaying serial dilutions of a CLuc containing sample (Figure 3), such as the culture medium of CLuc-expressing cells. In addition, the assay solution itself as well as the conditioned medium (culture medium from untransfected cells) should be included to establish the background in the assay.
    5. If excess activity for the instrument range is found, the sample should be diluted in either PBS or 10% serum-containing medium. The integration time can also be reduced, e.g. 2 seconds instead of 5 seconds.
    6. The presence of serum, i.e. culture medium, typically increases the background signal in the assay. For example, the CLuc assay solution alone shows 101–102 RLU; the CLuc assay solution in 10% FBS-containing medium results in 102–104 RLU, depending on the type of medium used.
    7. The recommended integration time for the luminescence measurement is 2–10 seconds. It is important to keep the integration time constant, in order to obtain consistent results.
    8. BioLux Cypridina Luciferase Assay Buffer (1X) and Substrate (lyophilized powder) must always be protected from light.
    9. BioLux Cypridina Luciferase Substrate is supplied in lyophilized form to ensure long shelf life and maximum stability.

    References

    1. Nakajima, Y. et al. (2004). Biosci. Biotechnol. Biochem. 63, 565-570.
    2. Yamagishi, K., Enomoto, T. and Ohmiya, Y. (2006). Anal. Biochemistry. 354, 15-21.
    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 save the unused portion of the CLuc assay solution?
    2. Does the activity of Cypridina Luciferase interfere with that of other luciferases such as Gaussia, Renilla & Firefly?
    3. What other reporter can I use along with Cypridina?
    1. Reconstitution of BioLux Cypridina Luciferase Substrate (E3309)
    2. Protocol I (Luminometers without injectors) (E3309)
    3. Protocol II (Injector-equipped luminometers) (E3309)

    Selection Tools

    Application Notes