Luciferase-based reporter-gene assays remain a useful and powerful method of high-throughput compound screening. However, false hits that result from direct interaction of compounds with the luciferase reporter can result in unnecessary follow-up efforts. The pNLCoI Vectors comprise a second-generation coincidence reporter vector system that allow expression of both firefly luciferase (luc2) and NanoLuc® Luciferase fused to a PEST destabilization domain (NlucP) from the same mRNA transcript. The stoichiometric expression of both luciferases is achieved by use of the P2A sequence from porcine teschovirus-1, which promotes a ribosomal skip and expression of the two unfused enzymes with distinct compound interaction profiles. When used in high-throughput compound screening, false hits caused by direct interaction with one or the other luciferases can be distinguished from true hits that show a similar response for both, reducing workload associated with follow-up screens.
The pNLCoI Vectors are designed for use with the Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay System, which allows sequential detection of firefly and NanoLuc® Luciferase in activity in the same sample. Both reagents provide stable glow-type luminescence signals with half-lives of approximately two hours allowing batch processing of samples and amenable to assays or screens in 96-, 384- or 1,536-well plate formats. Potent inhibition of firefly luciferase coupled with the high-intensity luminescence of NanoLuc® luciferase maximizes sensitivity for detection of both reporters.
Features – Benefits
- Improve Confidence and Save Time : Use of two different transcriptional reporters reduces false hit rates, increases the identification of true biological hits and eliminates time wasted on false-positive follow-up.
- Employ Robust and Sensitive Reporter Pair : luc2 and NlucP provide a bright reporter combination compatible with low-copy-number and plate scale-up, and provide greater signal-to-background compared to other reporters.
- Efficiently Identify False Hits : Firefly and NanoLuc® luciferase have dissimilar profiles of compound interference, enabling the identification of more false-positives than when either reporter is used alone.
- Use Simple Detection Format : Convenient “add-read-add-read” homogeneous format of NanoDLR™ assay is ideal for automation and HTS approaches.