MSDS
– For High-Throughput Applications
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GeneCopoeia’s Luc-Pair™ Duo-Luciferase HT Assay Kit is a convenient system for high-throughput detection of Firefly luciferase (FLuc) and Renilla luciferase (Rluc) activities in 96- or 384-well plates. The Luc-Pair™ Duo-Luciferase HT Assay reagents can be added directly to cells in growth medium without washing or preconditioning, and are optimized for use with many different media containing 0–10% serum (e.g. DMEM, RPMI1640, etc.) Firefly luciferase luminescence is produced by one reagent, while a second reagent simultaneously quenches the Firefly luciferase and produces Renilla luciferase luminescence.
The Luc-Pair™ Duo-Luciferase HT Assay Kit works great for many different eukaryotic cell types (e.g. mammalian, invertebrates, etc.) and is compatible with GeneCopoeia, Promega and many other Fluc and Rluc vectors.
Advantages
· Enhanced stability.
· Convenient — Directly lyse cells in culture medium and measure luciferase activities simultaneously.
· Versatility — Works well with many different eukaryotic (adherent or suspended) cells using micro-plate luminescence readers.
· Low background — Very limited background luminescence. No subtraction from readings is required.
· Simplicity — Renilla luciferase buffer contains the quenchers for Firefly luciferase activity (Figure 3). enabling a quick Glow and Stop-N-Glow two-step assays.
· Reproducibility — Reliable, linear results for a concentration range over several orders of magnitude.
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Figure 1. Activities of Firefly and Renilla luciferase signals using the GeneCopoeia (GCI) Luc-Pair Duo-Luciferase HT Assay Kit. HEK 293 cells were transfected with Promega pGL4.13/pGL4.75 reporter vectors for 48 hours. FLuc and RLuc activities were measured as described in the procedure. Promega’s Dual-Glo luciferase assay kit was used (FLuc-Prmg and RLuc-Prmg) in comparison.
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Figure 2. Firefly and Renilla luciferase activity mesured in various media. HEK 293 cells were transfected with Promega pGL4.13/pGL4.75 reporter vectors for 48 hours. 5×104 transfected cells were suspended in DMEM without serum or the following types of media containing 10% serum: DMEM, RPMI1640, EMEM, IMDM, McCoy’s 5A, F-12K, MEBM, ACL-4, L15, Cho-S-SFMII, NCTC109. Firefly and Renilla luciferase activities were measured as described in the procedure.
Figure 3. Firefly luciferase activity is quenched with the addition of Luc-HT Buffer II. HEK 293 cells were transfected with Promega pGL4.13/pGL4.75 reporter vectors for 48 hours. Firefly luciferase activity was measured as described in the procedure. Next, 1× Luc-HT Buffer II (without or with substrate) was added to the wells, followed by count reading in a luminometer. About 99.9% of firefly luciferase activity was quenched (middle column).