Flexstation ii384

FRET-based SUMO deconjugation assays were conducted by measuring the emission intensity of CyPet at 475 nm and YPet at 530 nm with an excitation wavelength of 414 nm in a fluorescence multiwall plate reader FlexStation II384 (Molecular Devices, Sunnyvale, CA, USA).
To test the substrate specificities, CyPet-SUMO1/2-RanGAP1C-YPet-GST was incubated with catalytic domains of SENP1/2/5/6/7 (1:1 molar ratio) at 37 °C in low salt reaction buffer (20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.1% (v/v) Tween-20 and 1 mM DTT) and transferred into a 384-well plate (glass bottom, Greiner, Monroe, NC, USA). The final concentration of reacted proteins was 100 nM. Reactions were stopped at 1 hr and were analyzed by fluorometer. Three samples were repeated in each condition. The results were reported as mean ± SD.
To study the differences of endopeptidase and isopeptidase activities of SENP1, CyPet-(pre-SUMO1/2)-YPet and CyPet-SUMO1/2-RanGAP1C-YPet-GST were incubated with SENP1C separately at 37 °C in low salt reaction buffer and transferred into a 384-well plate. The final concentrations of substrates and enzymes were 100 nM and 0.5 nM respectively. Reactions were tested within original 5 min with 10 s intervals. Initial velocities were derived by the developed method. Five samples were repeated in each concentration.

The FRET measurements were as previously described^{7 (link)}. Briefly, recombinant CyPet-SUMO1 and YPet-Ubc9 proteins were diluted with Tris buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl) in a total volume of 100 µL. For each set of measurements, the final concentrations of CyPet-SUMO1 were 0.5, 1.0 and 1.5 µM, respectively. The final concentrations of YPet-Ubc9 were increased from 0 to 4 µM. The fluorescence emission spectrum of each sample was determined using a fluorescence multi-well plate reader FlexstationII³⁸⁴ (Molecular Devices, Sunnyvale, CA). The fluorescence emission at 475 nm was measured at the excitation wavelength of 414 nm with a cutoff filter of 455 nm. For all the data points, the final fluorescent signals were obtained by subtracting the fluorescent signals with the background noise of blank well. The experiments were repeated three times and the average value of fluorescence were taken at each specific condition. The decrease of emission intensity at 475 nm (ΔEm₄₇₅) is calculated by subtracting the emission intensity at a specific YPet-Ubc9 concentration by the emission intensity of CyPet-SUMO1 only: $${\mathrm{\Delta }\text{Em}}_{475}={\mathrm{Em}}_{475\left(\left[\mathrm{YPetUbc}9\right]=\mathrm{X}\right)}-{\mathrm{Em}}_{475\left(\left[\mathrm{YPetUbc}9\right]=0\right)}$$

FRET-based SUMO processing assays were conducted by measuring the emission intensity of CyPet at 475 nm and of YPet at 530 nm with an excitation wavelength of 414 nm in a fluorescence multi-well plate reader (Flexstation II³⁸⁴, Molecular Devices, San Jose, CA, USA).
Recombinant CyPet-SUMO1-RanGAP1C-YPet substrate with different concentrations were incubated with recombinant 0.267 nM catalytic domain of SENP1 at 37 °C in buffer containing 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.1% (v/v) Tween-20, and 1 mM DTT to a total volume of 80 µL and added to each well of a 384-well plate (glass bottom, Greiner, Monroe, MI, USA). Reactions were tested within original 5 min. One phase association model was used to fit the exponential increased reaction velocity. Data were analyzed by the developed method and plotted in GraphPad Prism V software fitting the Michaelis–Menten equation. Five samples were repeated in each concentration.