| Assay Method Information | |
| | SARM1 NADase HPLC-Based Assay 2 |
| Description: | Reaction mixtures were prepared on ice by mixing 10 ul of SARM1 SAM-TIR cell lysate (320 fold dilution of lysate 11-3-2016, or 80 fold lysate dilution for assessment of time dependence) in PBS (pH 7.4) with 5 ul of compound stock. Compounds were first dissolved DMSO at 10 mM (final stock concentration). A 10 point compound dilution curve was prepared first with a 20 ul to 40 ul serial dilution in DMSO, followed by a 10 fold dilution (12 ul+108 ul) in PBS. Top concentration of compound in the assay is 250 uM. Compound and lysate were then preincubated, in duplicate, for various amounts of time (zero or 120 minutes for analysis of time dependence). 5 ul of 20 uM NAD+ (5 μM final concentration) was then added for a final reaction volume of 20 μl. The reaction was incubated at 37° C. for 60 (or 10 minutes @ room temp for assessment of time dependence), then stopped by addition of 180 μl of 0.55 M perchloric acid (HClO4). The reactions were then place on for 10 min, 16.6 μl of 3 M K2CO3 was added to neutralize the solution. Precipitated salts were removed by centrifugation 10 min at 4,000 rpm (Sorvall ST 16R centrifuge). 80 ul of the supernatant was analyzed by HPLC (Waters 2695) with Kinetex (50×4.6 mm, 5 μm; Phenomenex). NAD and metabolites were eluted with a 1 ml/min gradient from 100% A: KPO4 (5.026 g K2HPO4 and 2.876 KH2PO4 in 1 L H2O) to 3% methanol in 1 minute, followed by a linear gradient to 15% methanol in 1.5 minutes, held for 1 minute before returning to 0% methanol for 2.5 minutes for re-equilibration. NAD (3 minutes) and ADPR (1.5 min) were monitored by absorbance at 260 nm. Percent control conversion was established for each compound concentration. Blank (no lysate NAD only) values for ADPR were subtracted from samples and control (lysate+NAD) and control values from NAD depletion were subtracted from samples and blank to determine maximal ADPR conversion or NAD depletion (lysate dilutions used typically produced about a 50% conversion). Blanks and controls were run in triplicate (or more) then averaged. Duplicate data points from the 10 point dose curves were plotted using Grafit and IC50's were calculated using a 4 Parameter log fit. |
| Affinity data for this assay | |
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