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NanoBRET™ Signaling Protein Assays

N1880_NanoBRET--KRas-3aBRaf-Interaction-Asy_3

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This product is discontinued
NanoBRET™ Signaling Protein Assays
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Study Proteins in Their Natural Cellular Context

NanoBRET™ Signaling Protein Assays are sensitive, reproducible live cell assays designed for monitoring or screening the interaction of proteins involved in cell signaling events. Interactions between proteins are key events in normal cellular signal transduction pathways, and modulation of these interactions has been implicated in disease formation, making them important candidates for drug targeting. The NanoBRET™ KRas/BRaf Interaction Assay measures the specific interaction between mutant KRas (G12C) and BRaf human proteins in their natural cellular context. In epidermal growth factor receptor (EGFR) pathway-associated oncogenesis, mutations in KRas result in constitutive binding to BRaf even in the absence of growth factor, resulting in cell proliferation and suppressed apoptosis.

Better Separation Between Donor and Acceptor Signals

Energy Transfer from NanoLuc to HaloTag NanoBRET Ligand
Depiction of energy transfer from a NanoLuc®-Protein A fusion (energy donor) to a fluorescently labeled HaloTag®-Protein B fusion (energy acceptor) upon interaction of Protein A and Protein B. 
Donor and Acceptor Signal Wavelengths for NanoBRET
Spectral separation of the NanoLuc® emission (460nm) and the fluorescent HaloTag® NanoBRET™ 618 Ligand emission (618nm), and calculation of the NanoBRET™ ratio.

Notes

  • Sufficient NanoBRET™ Nano-Glo® Substrate and HaloTag® NanoBRET™ 618  Ligand are provided to perform 400 assays in 96-well plates or 1,000 assays in 384-well plates.
  • To perform NanoBRET™ PPI Assays an instrument, such as the GloMax® Discover, capable of sequentially measuring dual-filtered luminescence and equipped with appropriate filters is required.
  • The ideal filter setup will include a band pass (BP) filter centered around 460nm to measure the donor signal (e.g., Emission 450nm/BP 80nm) and a long pass (LP) filter starting at around 600–610nm to measure the acceptor signal (e.g., Emission 610nm/LP). Filters outside of these ranges will miss critical measurements and compromise data quality.
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Choose Starter Systems, Prebuilt or Custom NanoBRET™ Assays

Configure NanoBRET™ PPI Assays in a format that suits your needs. Build your own assays using the starter systems, choose from prebuilt assays in our catalog or consult with us to design a customized assay specific for your research.

Obtain High Assay Specificity

KRas-HaloTag and BRaf-NanoLuc NanoBRET DSA
Donor saturation assay performed by transfecting cells with a constant amount of BRaf-NanoLuc® donor DNA and increasing amounts of KRas(G12C)-HaloTag® acceptor DNA to represent increasing acceptor-to-donor (A:D) ratios. The negative control is membrane-localized NanoLuc® protein as a source of a mock donor in place of the BRaf-NanoLuc® fusion. The assay generates a hyperbolic curve, which indicates detection of a specific BRET interaction.
Signaling Protein Assay Vectors
12743MA-W
KRas(G12C)-HaloTag® Fusion Vector sequence & reference points.
12744MA-W
BRaf-NanoLuc® Fusion Vector sequence & reference points.
NanoBRET™ Control Vectors
12729MA-W
NanoBRET™ Positive Control Vector sequence & reference points.
12730MA-W
p53-HaloTag® Fusion Vector sequence & reference points.
12731MA-W
NanoLuc®-MDM2 Fusion Vector sequence & reference points.

Specifications

What's in the box?

Item Part # SizeAvailable Separately

HaloTag® NanoBRET™ 618 Ligand

G980A 2 × 20μl View Product

NanoBRET™ Nano-Glo® Substrate

N157A 2 × 50μl

p53-HaloTag® Fusion Vector

N159A 1 × 20μg

NanoLuc®-MDM2 Fusion Vector

N160A 1 × 20μg

KRas(G12C)-HaloTag® Fusion Vector

N177A 1 × 20μg

BRaf-NanoLuc® Fusion Vector

N178A 1 × 20μg

SDS

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Download SDSPDF (181 KB) – English (United States)

Certificate of Analysis

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Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

BB

Store at –20°C.

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Elisa

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