Product Class: Kit

BioLux® Cypridina Luciferase Starter Kit

Catalog #SizeConcentration
E3314S100 assays

Description

The BioLux Cypridina Luciferase Starter Kit contains a set of Cypridina Luciferase- expressing vectors and the reagents necessary for assaying the Cypridina Luciferase (CLuc) activity. The pSV40-CLuc Control Plasmid is a mammalian expression vector encoding the secreted Cypridina Luciferase from the Ostracod Cypridina noctiluca (1,2) under the control of the constitutive SV40 promoter. pSV40-CLuc can be used as a control to determine transfection efficiency. The pCLuc-Basic 2 Vector lacking promoter elements is a cloning vector for mammalian expression. pCLuc-Basic 2 contains a multiple cloning site (MCS) upstream of the CLuc coding sequence. In addition, the neomycin resistance gene in the pCLuc-Basic 2 cloning vector allows selection for stable integration of the plasmid into the mammalian cell genome.

Cypridina Luciferase is a 62 kDa protein with a native signal peptide at the N-terminus allowing it to be secreted from mammalian cells. This luciferase does not require ATP and catalyzes the oxidation of its luciferin substrate in a photochemical reaction (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). 
  • Secreted CLuc is thermally stable at 55°C (Figure 3A), 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 3B). 
  • The CLuc assay is very sensitive, allowing detection of very small amounts of Cypridina Luciferase activity (Figure 4). 
  • 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).

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: 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 4: Sensitivity of Cypridina Luciferase assay.
The CLuc activity was assayed from the 10-fold serial dilutions of the supernatant from a stable CLuc-expressing cell line.
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.
Features of pSV40-CLuc
  • Polylinker MCS: 1–52 
  • SV40 promoter: 52–247 
  • CLuc ORF: 292–1953 
  • Start codon of CLuc: 292–294 
  • Stop codon of CLuc: 1951–1953 
  • Signal peptide: 292–345 
  • SV40 poly-A site: 1968–2189 
  • SV40 enhancer: 2196–2442 
  • Bacterial replication ori (pMB1): 3348–2760 
  • Ampicilin resistance gene: 4379–3529
Features of pCLuc-Basic 2
  • Polylinker MCS: 20–68 
  • Start codon of CLuc: 75–77 
  • Stop codon of CLuc: 1734–1736 
  • Signal peptide: 75–128 
  • Synthetic poly-A site: 1745–1793 
  • neoR (SV40) promoter: 2379–2714 
  • Neomycin resistance gene: 2766–3560 
  • Bacterial replication ori (pMB1): 4894–4306 
  • Ampicilin resistance gene: 5925–5065
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

Supporting Documents

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].

Notes

  1. Four recommended sequencing primers for pCLuc-Basic 2 Vector are as follows:

    • pBasic Forward Sequencing Primer (23-mer) (not available at NEB)
      5´-GGGGTTCCGCGCACATTTCCCCG-3´ (6020–6042) 
    • pBasic Reverse Primer (25-mer) (not available at NEB)
      5´-TCAGAAGCCATAGAGCCCACCGCAT-3´ (1888–1864) 
    • CLuc 3´ End Forward Primer (23-mer) (not available at NEB)
      5´-GAGTTCAAGAAAGAATGCTACAT-3´ (1671–1693)
    • CLuc 5´ End Reverse Primer (24-mer) (not available at NEB)
      5´-GTAAGGACAGTCCTGGCAATGAAC-3´ (143–120)
  2. For improving the transfection efficiency and transfecting difficult-to-transfect cell lines (suspension cells, primary cells, monocytes, etc.), we recommend using TransPass V (NEB #M2561) in combination with TransPass D2 (NEB #M2554) or TransPass D1 (NEB #M2553). For more information on the TransPass Transfection Reagent products, please go to the product page.
  3. Constructs made from the pCLuc-Basic vector can be used to create stable clonal cell lines using G418 selection (refer to Transfection Protocol II).
  4. G418 (Neomycin) should be included in the complete growth media when culturing a stable clonal cell line.
  5. Store the BioLux Cypridina Luciferase Assay Buffer (1X) at -20°C for up to 1 year.
  6. The BioLux Cypridina Luciferase Assay Buffer (1X), the reconstituted CLuc substrate (100X solution) and the CLuc assay solution must be protected from light.
  7. 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.
  8. 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.
  9. 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.
  10. 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.
  11. The 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).
  12. BioLux Cypridina Luciferase and Substrate (lyophilized powder and substrate solution) must always be protected from light.
  13. 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.
  1. Transfection Guidelines (E3314)
  2. Reconstitution of BioLux Cypridina Luciferase Substrate (E3314)
  3. CLuc Activity Assay Protocol I (Luminometers without injectors) (E3314)
  4. CLuc Activity Assay Protocol II (Injector-equipped luminometers) (E3314)

Application Notes