Nano-Glo® Dual-Luciferase® Reporter Assay

Greater Ease-of-Use and Workflow Flexibility Your Choice of Two Primary Reporters Greater Sensitivity at Low Expression Levels

The Nano-Glo® Dual-Luciferase® Reporter (NanoDLR) Assay allows you to detect the activities of firefly and NanoLuc® luciferases in a single sample. Improved firefly chemistry and the bright, sensitive NanoLuc® luciferase make this a highly sensitive dual assay that is simple to use, provides superior data quality, and allows greater flexibility in assay design.

Switch Seamlessly from Your Current Dual-Luciferase® Assay!

The Nano-Glo® Dual-Luciferase® Reporter Assay uses the same protocol as the popular Dual-Glo® Luciferase Assay, with greatly improved sensitivity, performance and convenience.

Easy ways to get started with the NanoDLR Reporter Assay:

  • Choose a free NanoLuc® or firefly luciferase control vector with a TK or PGK promoter by ordering a Nano-Glo® Dual-Luciferase® Reporter Assay/vector bundle.

    Order Reagent + Vector Bundle

  • Skip a cloning step by choosing an optimized response element or promoter-containing vector to study your pathway of interest using either the NlucP or luc2P reporter.

    View Signaling Pathway Vectors

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.

NanoDLR Reporter Assay give brighter signals with stable signal kinetics.

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 in the NanoDLR Assay.

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.

Sensivity of NanoLuc Lucifersase in the NanoDLR Assay compared to DLR and Dual-Glo Assays.

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, the brightest most responsive dual-assay format.

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

Firefly luciferase primary reporter and NanoLuc control, a bright sensitive control reporter.

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 with NanoLuac and Firefly luciferase reporters.

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).

Express firefly and NanoLuc luciferase reporters from a single open reading frame in a coincidence assay.

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.

Improved NanoDLR reagent stability.

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.
GloMax Discover Multimode Reader