A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays

doi:10.1016/j.ab.2006.04.046

Analytical Biochemistry

Volume 356, Issue 1, 1 September 2006, Pages 94-99

https://doi.org/10.1016/j.ab.2006.04.046 Get rights and content

Abstract

We established a quantitative reporter gene protocol, the P/Rluc assay system, allowing the sequential measurement of Photinus and Renilla luciferase activities from the same extract. Other than comparable commercial reporter assay systems and their noncommercial counterparts, the P/Rluc assay system was formulated under the aspect of full compatibility with standard methods for protein assays. This feature greatly expands the range of applications for assay systems quantifying the expression of multiple luciferase reporters.

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Tissue culture and plasmids

HeLa cells (ATCC CCL-2) were cultured at 37 °C with 5% CO₂ in minimal essential medium (Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum (Biochrom, Berlin, Germany), 2 mM l-glutamine, 50 μg/ml penicillin, and 50 μg/ml streptomycin (all from Invitrogen, Carlsbad, CA, USA). For transient transfections, the Roti-Fect liposome formulation (Carl Roth, Karlsruhe, Germany) was used according to the manufacturer’s protocol. Expression vectors for Photinus and Renilla luciferase genes will be

Formulation of a lysis solution

A P/Rluc assay system meeting the requirements for full compatibility with standard protein assays necessitates the formulation of a lysis solution other than PLB. We tested some commonly used cell lysis detergents in PPB. This buffer itself triggered coelenterazine autoluminescence levels only marginally above those measured for water (Table 1). In contrast, Triton X-100 and NP40 caused a very substantial, concentration-dependent increase of coelenterazine autoluminescence. For other

Discussion

With its widespread use, the DL assay can be considered the “gold standard” for the sensitive and accurate quantification of Pluc and Rluc in a single assay setup. Indeed, its quenching of Pluc activity is slightly more efficient when compared with the P/Rluc assay system introduced here. Other quantitative parameters are similar between the two systems. The ncDL system is somewhat less efficient. However, that assay system also is perfectly suited to guarantee accurate quantification of both

Acknowledgments

We thank Gesine Gunkel for excellent technical assistance and the members of our laboratory for continuous helpful discussions. Daniel Besser and Mathias Bell are acknowledged for critically reading the manuscript. The work was supported by a grant from the Volkwagen Stiftung to M. Gossen.

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A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays

Abstract

Section snippets

Tissue culture and plasmids

Formulation of a lysis solution

Discussion

Acknowledgments

Anal. Biochem.

J. Biol. Chem.

Anal. Biochem.

Gene

Arch. Biochem. Biophys.

Anal. Biochem.

Arch. Biochem. Biophys.