The sensitivity of split NanoLuciferase and split GFP was assessed by combining a fixed amount of purified LgBiT or GFP1–10 protein with increasing molar equivalents of HiBiT or GFP11 peptide. For split NanoLuciferase, 25 µL of desired concentrations (half-log increases) of HiBiT peptide were combined with 50 µL of 100 nM LgBiT protein in a black 96-well clear bottom microplate. To the mixture, 25 µL of NanoGlo Live Cell Substrate (Promega) was added and luminescence was measured on the IVIS Lumina II (Perkin Elmer) 10 min after substrate addition. Luminescence data were processed with Living Image 4.3.1 software. Luminescence was quantified in average radiance in units of photons per second per centimetre squared per steradian (p/s/cm2/sr).
For split GFP, 50 µL of desired concentrations of GFP11 peptide (GL BioChem) was combined with 50 µL of 6 µM GFP1–10 for a final volume of 100 µL in a black 96-well clear bottom microplate. Incubation proceeded at 37 °C inside ClarioSTAR microplate reader (BMG Labtech) with fluorescence emission at 515 nm being detected after 470 nm excitation 2.75 h post GFP11 addition (the time at which peak fluorescence intensity was observed).
Radiance and fluorescence values recorded were averages of three experiments subtracted by the average radiance or fluorescence values of blank media or PBS.
For split GFP, 50 µL of desired concentrations of GFP11 peptide (GL BioChem) was combined with 50 µL of 6 µM GFP1–10 for a final volume of 100 µL in a black 96-well clear bottom microplate. Incubation proceeded at 37 °C inside ClarioSTAR microplate reader (BMG Labtech) with fluorescence emission at 515 nm being detected after 470 nm excitation 2.75 h post GFP11 addition (the time at which peak fluorescence intensity was observed).
Radiance and fluorescence values recorded were averages of three experiments subtracted by the average radiance or fluorescence values of blank media or PBS.