Synergi 2.5 mm fusion rp 100a 10033 0 mm lc column

Each reaction contained 25 mM HEPES pH 7.4, 10 mM KCl, 10 mM MgCl 2 , 1 mM TCEP, 25 nM MBP SARM1 409-724 , and NAD starting at 100 μM and serially diluted two-fold for six total concentrations. Compound was added at 0, 40 or 200 nM in DMSO, and reactions were incubated at room temperature for 0, 5, 10 and 15 min. to determine initial reaction velocities (v 0 ). Reactions were run in duplicate. After the incubation times, samples were quenched with an equal volume of 0.4% formic acid. Samples were run using an Agilent HPLC 1260 Infinity II with a Synergi 2.5 mM Fusion-RP 100A° (10033.0 mm) LC column from Phenomenex. Total run time for each sample was 4 min. The run was isocratic with 1.5% methanol in 40 mM ammonium acetate pH 6.0. Samples were run at a flow rate of 0.8 mL/min at 55°C. Peak areas of NAM were determined using OpenLab CDS ChemStation Edition software (Agilent). To determine kinetic parameters, NAD concentration was plotted versus v 0 and data was fit using a Michaelis Menten fit in Prism 9.

Each reaction contained 25 mM HEPES pH 7.4, 10 mM KCl, 10 mM MgCl 2 , 1 mM TCEP, and 5 nM SARM1 409-724 or SARM1 50-724 with 20 or 200 mM NAD. For SARM1 50-724 , autoinhibition was released by addition of 0.3 mM NMN. For SARM1 559-700 , higher concentrations of up to 15 mM were used. For dose-response of NAM, NAM was added to reactions starting at a final compound concentration of 50 mM and diluted serially 1:3. The reactions were incubated at room temperature for 60 min and quenched with an equal volume of 0.4% formic acid. The samples were run on Agilent HPLC 1260 Infinity II with Synergi 2.5 mM Fusion-RP 100A ˚(100 3 3.0 mm) LC column from Phenomenex. Total run time for each sample was 4 min. The run was isocratic with 1.5% methanol in 40 mM ammonium acetate pH 6.0. Samples were run at a flow rate of 0.8 mL/min at 55 C. Peak areas of ADPR and NAM were determined using OpenLab CDS ChemStation Edition software (Agilent).