Fluorolog 3 22 photon counting spectrofluorometer

iTFS spectral measurements were performed in a 4 × 4 mm quartz cuvette (Starna Cells, Inc., Atascadero, CA) at 25°C using a Fluorolog 3–22 photon-counting spectrofluorometer (Horiba Jobin Yvon) equipped with a 450-W xenon lamp, double-excitation and double-emission monochromators, a cooled PMT housing, and a temperature-controlled cuvette compartment. WT Drp1, CT+, and site-directed mutants were diluted to 0.5 μM final in buffer A containing 1 mM DTT. Buffer background- and instrument-corrected Trp fluorescence spectra were obtained by selectively exciting Trp at 295 nm (2-nm bandpass) and emission monitored at 1-nm increments between 315 and 415 nm (2-nm bandpass). Data are averages of three scans for each sample.

iTFS measurements were performed in a 4 × 4 mm quartz cuvette (Starna Cells, Inc., Atascadero, CA) at 25 °C using a Fluorolog 3-22 photon-counting spectrofluorometer (Horiba Jobin Yvon) equipped with a 450-W xenon lamp, double-excitation and double-emission monochromators, a cooled PMT housing, and a temperature-controlled cuvette compartment. WT Drp1, CT + , and site-directed mutants were diluted to 0.5 µM final in buffer A containing 1 mM DTT. Buffer background- and instrument-corrected Trp fluorescence spectra were obtained by selectively exciting Trp at 295 nm (2-nm bandpass) and emission monitored at 1-nm increments between 315 and 415 nm (2-nm bandpass). Data are averages of three scans for each sample.

Drp1 WT and mutants at 1 μM each in a total final volume of 400 μl were equilibrated in Buffer A containing 2 mM MgCl₂ and 1 mM DTT in a 4 mm × 4 mm quartz cuvette (Starna cells, CA) temperature-equilibrated at 25 °C. Scattered light from these samples was measured continuously at 350 nm (0.5 nm bandpass; 0.5 sec integration time) using a Fluorolog 3–22 photon-counting spectrofluorometer (Horiba, NJ), before and after addition of GMP-PCP or GTP to a final concentration of 1 mM (marked by arrows) at a defined time point. The intensity traces were corrected for dilution upon addition of nucleotide.