Nano-Glo® HiBiT Dual-Luciferase® Reporter System

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Multiplexed Detection of HiBiT-Tagged Proteins and Firefly Luciferase in the Same Sample

  • Rapidly identify specific and non-specific effects of compounds
  • Compatible with batch processing and automation
  • Separate and bicistronic systems for co-expression of HiBiT and Fluc available

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Nano-Glo® HiBiT Dual-Luciferase® Reporter System
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Sequential Measurement of Firefly Luciferase and HiBiT-Tagged Proteins from the Same Sample

The Nano-Glo® HiBiT Dual-Luciferase® Reporter System (HiBiT NanoDLR™) allows sequential measurement of firefly luciferase (Fluc) and HiBiT-tagged proteins from the same sample in an “add-read-add-read” assay format. Fluc is used as a constitutively expressed control to indicate nonspecific compound effects on cell viability or protein expression, rather than specific changes in the levels of the HiBiT-tagged protein of interest, aiding in the interpretation of protein modulation screens and studies.

The 11-amino acid HiBiT peptide tag can be added to a protein of interest using CRISPR/Cas9 gene editing coupled with ectopic expression of Fluc. Alternatively, use a bicistronic HiBiT entry vector to express both the HiBiT-protein fusion and constitutive Fluc on the same mRNA.

HiBiT NanoDLR™ Workflow

18324ma - HiBiT NanoDLR™ workflow

Firefly luciferase (Fluc) is first detected after addition of the ONE-Glo™ EX reagent supplemented with LgBiT (1). The NanoDLR™ Stop & Glo® Reagent is added to the cells to both quench the firefly signal and detect the now-complemented NanoBiT® (HiBiT + LgBiT) signal (2).

Use the Fluc Signal to Distinguish Specific and Nonspecific Compound Effects on Protein Levels

18325ma-a - Selective HiBiT-BRD4 degradation with MZ1
18325ma-b - Selective HiBiT-BRD4 degradation with dBET1
18325ma-c - General cellular toxicity with staurosporine

Selective HiBiT-BRD4 degradation with two PROTACs compared to general cellular toxicity. HEK293 cells were transiently transfected with a construct expressing the HiBiT-BRD4-IRES-luc2 cassette from the weak TK promoter. Cells were treated with titrating concentrations of MZ1 (Panel A), dBET1 (Panel B) or staurosporine (Panel C) before the Fluc (blue) and HiBiT (red) signals were measured using the lytic HiBiT NanoDLR™ Assay. MZ1 and dBET1 selectively link BRD4 to two different E3 ligase complexes for targeted degradation, and staurosporine causes general toxicity, resulting in loss of both HiBiT-BRD4 and firefly luciferase (Fluc).

Quantifiable Over Seven Orders of Magnitude

18326ma - Background-subtracted RLU from firefly luciferase in ONE-Glo EX/LgBiT reagent
18327ma - Background-subtracted RLU from HiBiT in NanoDLR Stop & Glo Reagent

The broad linear dynamic range accurately quantifies tagged proteins regardless of expression level to measure changes in protein abundance. The HiBiT NanoDLR™ Assay can quantify even low-abundance proteins at endogenous levels of expression.

Express HiBiT and Fluc on the Same mRNA with Bicistronic Vectors

18328ma - Create N- or C-terminal HiBiT fusion vectors and express from different promoter strengths (CMV, PGK or TK)

A variety of bicistronic HiBiT entry vectors with constitutive expression of firefly luciferase are available to create N- or C-terminal HiBiT fusion vectors and express from different promoter strengths (CMV, PGK or TK). All are compatible with stable cell line generation with a blasticidin selection marker.

Patents and Disclaimers

Materials may be covered by pending or issued patents or may have certain limitations.
Information is available upon request and will be included on applicable quotes.

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