Fluorescence-Based SUMO Conjugation Assay

All components of the SUMOylation assay (0.5 mM CyPet-SUMO1, 0.1 mM E1, 0.2 mM E2, 0.25 mM E3 PIAS1, and 2 mM YPet-Linker3-M1) were combined in the SUMOylation buffer containing 50 mM Tris-HCl pH 7.4, 1 mM DTT, and 4 mM MgCl₂ in a total volume of 60 mL. The sample mixtures were an added 1 mM ATP were incubated in an Eppendorf tube at 37 °C, and all sample mixtures were transferred to the Greiner 384-well plate (Sigma-Aldrich). The fluorescence emissions were measured using FlexstationII384 (Molecular Devices, Sunnyvale, CA, USA). Emission intensities were measured at three wavelengths: 475 and 530 nm after excitation at 414 nm, and 530 nm after excitation at 475 nm [42 (link)].