NanoDLR™ Assay Gives Bright, Stable Signals
In the Nano-Glo® Dual-Luciferase® Reporter Assay, firefly luciferase activity is measured first using the ONE-Glo™ EX Luciferase Assay Reagent. NanoDLR™ Stop & Glo® Reagent is then added to quench the firefly signal and provide the substrate needed to measure NanoLuc® luciferase activity. The signal half-life is greater than two hours for both reporters.
Brighter signals with stable signal kinetics. Comparison of luminescence signals from HEK293 cells transfected with a 1:1:8 ratio of either TK-Rluc (Renilla):TK-Fluc (firefly):carrier DNA or TK-Nluc (NanoLuc):TK-Fluc:carrier DNA and assayed with NanoDLR™, DLR™ or Dual-Glo® Dual-Luciferase Assay System as indicated. When luciferases are expressed from the same promoter, the NanoDLR™ Assay provides bright signal for both Fluc and Nluc reporters with stable "glow-type" signals.
Better Quenching, Greater Sensitivity
Potent inhibition of firefly luciferase (Fluc) provides excellent signal separation, allowing accurate measurement of even low amounts of NanoLuc® luciferase (Nluc), even in the presence of high Fluc activity.
Efficient quenching of firefly luciferase signal. Even extremely high Fluc signals are reduced to near background levels by NanoDLR™ Stop & Glo® Reagent, providing excellent signal separation and low variability without sacrificing the sensitivity of two bright luminescent readouts.
Greater Sensitivity of NanoLuc® Luciferase Compared to Renilla in Dual-Glo® and DLR™ Assays
The bright NanoLuc® signal and improved quenching of the firefly luciferase signal provide greater sensitivity and improved signal:background ratio. This superior quenching, coupled with the high-intensity luminescence of NanoLuc® luciferase, maximizes sensitivity for detection of both reporters.
Nanoluc® Luciferase sensitivity. Luminescence from a titration of purified Renilla or NanoLuc® luciferase was compared in the presence or absence of a high concentration (100nM) of purified firefly luciferase. Sensitivity was determined as the amount of enzyme necessary to achieve a signal double the background luminescence (dashed lines).
In the absence of firefly luciferase, the glow-type NanoLuc® signal is several-fold more sensitive than the flash-type Renilla signal of the DLR™ Assay. In the presence of a high firefly signal, the NanoDLR™ Assay is nearly 100-fold more sensitive.
The NanoDLR™ Assay was >300-fold more sensitive than the Dual-Glo™ Assay in the absence of firefly luciferase, and >3000-fold more sensitive in the presence of firefly luciferase.
Use Either Firefly or NanoLuc® Luciferase as the Primary Reporter or use Both as Dynamic Reporters
The Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay, can be used in different configurations to suit your assay design.
NanoLuc® Luciferase as Primary Reporter
The brightest, most responsive format.
Best for: Low cell numbers, plate scale-up or challenging cell lines.
Reporter Constructs: NlucP Experimental Reporter; Fluc Control
NanoLuc® luciferase primary reporter. Highlighted data shows luminescence values for HEK293 cells transfected with NFkB-NanoLuc® PEST vector and TK-Fluc control at 10:1 ratio. Cells were treated with TNFα and reporter activity measured using the NanoDLR™ assay 4 hours post-treatment.
Firefly Luciferase as Primary Reporter
Use existing firefly luciferase constructs, and add NanoLuc® luciferase for a more robust control.
Best for: Standard reporter assay formats. The NanoLuc® luciferase control reporter is up to 1000X brighter than Renilla luciferase, so you can use less control DNA.
Reporter Constructs: FlucP Experimental Reporter; Nluc Control
NanoLuc® luciferase control reporter. Highlighted data shows luminescence values for HEK293 cells transfected with a NFkB response element Fluc-PESTvector and a TK-Nanoluc® control vector at 10:1 ratio. Cells were treated with TNFα and reporter activity measured using the NanoDLR™ assay 4 hours post-treatment.
Two Primary Reporters
Measure 2 different biological responses simultaneously.
Best for: Measuring 2 pathways at once for maximum data; multiplexing protein stability and genetic reporter assays.
Reporter Constructs: NlucP Experimental Reporter; FlucP Experimental Reporter; Nluc protein stability reporter.
Measure protein dynamics and gene activation in the same sample. HEK293 cells were transiently transfected with the pNLF1-HIF1A[CMV/neo] fusion construct, diluted 1:1000 into the Hypoxia Response Element Vector, pGL4.42[luc2P/NRE/Hygro]. After 18 hours, cells were stimulated with varying doses of 1,10-phenanthroline and assayed for expression of the firefly luciferase transcriptional reporter and the HIF1a-NanoLuc® fusion protein using the Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay at the indicated time points.
Coincidence Reporter System
Express both reporters from a single open reading frame.
Best for: Distinguishing false hits in HTS from compounds truly affecting expression of target gene.
Reporter Construct: Firefly-P2A-NanoLuc®-PEST construct (pNLCoI Vectors).
Improved Reagent Stability Simplifies Repeat Use and Reduces Waste
The reconstituted ONE-Glo™ EX Reagent in the NanoDLR™ Assay has increased stability at room temperature and 7°C , reducing the need to aliquot and freeze reagent between uses.
The NanoDLR™ Reagents display extended stability once reconstituted. Reconstituted reagents were stored at either 22°C or 7°C and frozen at –70°C at defined times. Upon thawing, reagents were combined with purified firefly or NanoLuc® luciferase and the relative activity calculated as the luminescence signal intensity for each sample measured 3 minutes after enzyme addition, relative to the signal intensity of the sample that was placed at – 70°C with no 22°C incubation; n=3.
Compatible with Many Workflow Formats to Support Any Throughput Scale
The extended signal stability of the Nano-Glo® Dual-Luciferase® Reporter Assay enables an easy, add-mix-read assay protocol. Reagents can be used with injection-based protocols or with cell lysates made with passive lysis buffer.
- Homogeneous Assay Format: Assay cells directly in growth medium. A two-hour signal half-life for both reporters supports batch addition of reagents to multiple plates.
- Injection-based Assay Format: Deliver reagents to cells using luminometers, such as GloMax® Discover, equipped with dual injectors or with bulk reagent dispensers.