Application note: Performing NanoBRET with the Varioskan LUX multimode reader

In this paper, Thermo Fisher Scientific aim to assess the performance of Thermo Scientific Varioskan LUX when running NanoBRET.

NanoBRET technology introduced by Promega is designed for studying protein-protein interaction (PPI). In NanoBRET, PPI can be studied with a new efficient donor-acceptor pair.

The new pair consists of an extremely bright blue-shifted luciferase (NanoLuc) as energy donor and a fluorescently labelled red-shifted HaloTag fusion protein as energy acceptor. The new donor (NanoLuc) has a small size (19kDa), very high emission intensity (460nm emission peak) and a relatively narrow spectrum, which reduces the chances for donor signal ‘bleeding’ into the acceptor emission channel. In turn, the selection of a red-emitting fluorescent NanoBRET ligand (618nm emission) that covalently attaches to HaloTag as an acceptor molecule allows for a considerable spectral separation (over 150nm), thus ensuring very high signal-to-background ratios. However, performance still depends on the selection of a suitable microplate reader.