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