BioLux® Cypridina Luciferase Starter Kit

The assay reagents and the plasmids are available for purchase as stand-alone products (E3309S/L, N0317S, & N0318S).

  • This product was discontinued on 01/05/2015

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    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.
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