Announcement: Aurelia Bioscience has been acquired by Charnwood Molecular.

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Important information: Aurelia Bioscience website pages now redirect to Charnwood Molecular following the company's acquisition. If you have any questions about our bioscience services or specific content you are looking for please contact info@charnwood-molecular.com

Introduction
Promega’s NanoBRET™ TE kinase assay measures compound binding to full-length kinase targets in living cells. This quantitative analysis is based on Bio-Resonance Energy Transfer (BRET) between photons generated by a highly efficient luminescence enzyme, Nanoluc, and a cell permeable fluorophore tagged to a compound (tracer) that binds specifically to the kinase in living cells.

Advantages

  • The first biophysical technique to profile compound binding for intracellular targets, i.e. a functional pharmacology assay
  • Allows intracellular examination of compound binding to the target kinase at physiological ATP concentrations in living cells
  • Allows expression of full-length kinase target in cells
  • Clinically relevant mutated kinase sequences can be expressed in cells to examine changes in the binding properties of compounds
  • Cells can be disrupted (with digitonin) to allow compound access in the absence of a competent cell membrane to performed the assay in a pseudo biochemical binding format
  • Allows examination of the compound K-on (association rate) and K-off (dissociation rate) with kinases in living cells
  • The approach can be used to look at allosteric and ATP competitive inhibitors
  • How it works
    A cDNA construct consisting of the full-length kinase cDNA and Nanoluc luciferase enzyme cDNA are transiently expressed in cells (lipofection followed by overnight incubation at 37°C / 5% CO2) to generate a chimeric protein. Media is replaced with assay buffer containing the cell permeable Nanoluc substrate, generating photons that are detected in the fluorescence microplate reader at 460nm.

    The tracer, which is a pan agonist labelled with a fluorophore, is added and binds to the kinase such that the fluorophore is brought into close proximity with the Nanoluc enzyme. This triggers the activation of the fluorophore via a process known as Bio Resonance Energy Transfer (BRET). The fluorophore emits at 610nm and this signal is measured within the fluorescence microplate reader.

    The BRET response is a ratio of the signal at 610nm divided by the signal at 460nm. Both the tracer and the Nanoluc substrate are added simultaneously to the cells. The addition of test compounds results in competition for binding to the kinase, and the tracer is competed off the kinase, moving the fluorophore away from the 460nm photon source and thus decreasing the BRET signal at 610nm.

    This process is a competition assay between the compound and the tracer for binding to the kinase allowing quantification of the intracellular affinity of the test compound within living cells at physiological pH and ATP concentration.