HEK293T seeded in six-well plates were transfected with 50 ng of donor plasmid (RLuc8-KRASG12D-CAAX) and 100–150 ng of acceptor plasmid (RAF1RBD-GFP2, PI3KαRBD-GFP2, PI3KγRBD-GFP2, RALGDSRA-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.
2103 multilabel reader
Manufactured by PerkinElmer
The 2103 Multilabel Reader is a versatile lab instrument designed to perform a wide range of absorbance, fluorescence, and luminescence measurement applications. It offers a broad detection range and flexible configuration options to support various assay types and microplate formats.
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5 protocols using 2103 multilabel reader
1
BRET2 Assay for Ras-Effector Binding
2
BRET2 Biosensor Measurement Protocol
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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.
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.
3
Optimizing Bacterial Growth Conditions
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Unless otherwise stated, liquid bacterial cultures were grown in 300 μL of 2×YT (casein peptone 16 g/L, yeast extract 10 g/L, NaCl 5 g/L) supplemented with potassium phosphate (50 mM, pH 7), referred to hereafter as “media”, and incubated at 37 °C in 48-well flat-bottomed plates (CELLSTAR, Greiner Bio-One) with shaking at 200 rpm. Solid growth media was prepared with 2% agar. Antibiotics were used, as appropriate, at the following concentrations: ampicillin, 100 μg/mL; zeocin, 50 μg/mL; chloramphenicol, 5 μg/mL. All selective agents were purchased commercially. Stock solutions of N6-[(2-propynyloxy)carbonyl]-L-lysine HCl (PrK; SC-36420, Synchem) and p-azido-L-phenylalanine HCl (pAzF; 06162, Chem-Impex International) were dissolved in ddH2O (1 M PrK) or 0.2 M NaOH (250 mM pAzF), respectively. Cell growth was measured using a Perkin Elmer Envision 2103 Multilabel Reader with a 590/20 nm filter. sfGFP fluorescence was measured using a Perkin Elmer Envision 2103 Multilabel Reader with a 485/14 nm filter for excitation and 535/25 nm filter for emission. The fluorescence of the media used in each experiment was measured and subtracted from the fluorescence measurements of each cell culture.
4
Bioluminescence Resonance Energy Transfer Assay
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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.
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.
5
Optimizing Bacterial Growth Conditions
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