Bret2 dual emission optical module

HEK293T seeded in six-well plates were transfected with 50 ng of donor plasmid (RLuc8-KRASG12D-CAAX) and 100–150 ng of acceptor plasmid (RAF1_RBD-GFP2, PI3Kα_RBD-GFP2, PI3Kγ_RBD-GFP2, RALGDS_RA-GFP2) and with 100 ng of appropriate competitor plasmid (pEF-DARPin-myc). Cells were detached 24 h later and seeded in a white 96-well plate (clear bottom, PerkinElmer, cat#6005181). Cells were left for an additional 24 h at 37 °C. The BRET2 signal was read directly after addition of Coelenterazine 400a substrate (10 μM final) on cells (Cayman Chemicals, cat#16157) on an Envision instrument (2103 Multilabel Reader, PerkinElmer) with the BRET2 Dual Emission optical module (515±30 nm and 410±80 nm; PerkinElmer). The experiment shown in Fig. 6 was performed with technical triplicates and also repeated once with similar results.

BRET2 signal was determined immediately after addition of coelenterazine 400a substrate (10 μM final) to cells (Cayman Chemicals), using an Envision instrument (2103 Multilabel Reader, PerkinElmer) with the BRET2 Dual Emission optical module (515 nm – 30 nm and 410 nm – 80 nm; PerkinElmer). Total GFP² fluorescence was detected with excitation and emission peaks set at 405 nm and 515 nm, respectively. Total RLuc8 luminescence was measured with the Luminescence 400–700 nm-wavelength filter.
The BRET signal or BRET ratio corresponds to the light emitted by the GFP² acceptor constructs (515 nm – 30 nm) upon addition of coelenterazine 400a divided by the light emitted by the RLuc8 donor constructs (410 nm – 80 nm). The background signal is subtracted from that BRET ratio using the donor-only negative control where only the RLuc8 plasmid is transfected into the cells. The normalized BRET ratio is the BRET ratio normalized to a negative control (DMSO, no competitor or iDAb control) during a competition assay. Total GFP² and RLuc8 signals were used to control the protein expression from each plasmid.

BRET2 signal was determined immediately after addition of coelenterazine 400a substrate (10 μM final) to cells (Cayman Chemicals), using an Envision instrument (2103 Multilabel Reader, PerkinElmer) with the BRET2 Dual Emission optical module (515–30 and 410–80 nm; PerkinElmer). The total GFP² fluorescence was detected with excitation and emission peaks set at 405 and 515 nm, respectively. The total mCherry fluorescence was detected with excitation and emission peaks set at 530 and 615 nm, respectively. The total RLuc8 luminescence was measured with the luminescence 400–700 nm-wavelength filter.
The BRET signal or BRET ratio corresponds to the light emitted by the GFP² acceptor constructs (515–30 nm) upon addition of coelenterazine 400a divided by the light emitted by the RLuc8 donor constructs (410–80 nm). The background signal is subtracted from that BRET ratio using the donor-only negative control, where only the RLuc8 plasmid is transfected into the cells. The normalised BRET ratio is the BRET ratio normalised to a negative control (DARPin control) during a competition assay. The total GFP², mCherry and RLuc8 signals were used to control the protein expression level from each plasmid.