Ammonium formate

Two assays were used to further validate HTS hits and further remove fluorescence interfering compounds from consideration. C12-NBD ceramide (Cayman chemical) was used as a fluorescent nCDase substrate and reaction products separated by reverse phase HPLC. Reaction conditions were 20 μM substrate, 1 nM nCDase in 75 mM NaCl, 25 mM HEPES (pH 8.0), 0.4% Triton X-100, for 2 hours at 37°C in a final volume of 100 μL. Samples were extracted with 1:1 chloroform-methanol (Sigma), dried under nitrogen gas, and resuspended in 60 μL HPLC mobile phase B. Reaction products were separated by reverse phase HPLC using a Spectra 3um C8SR column (3 μm particle, 3.0 × 150 mm, Peeke Scientific). Mobile phase A contained 0.2% formic acid, 1 mM ammonium formate in HPLC grade water (Sigma). Mobile phase B contained 0.2% formic acid (Fisher), 1 mM ammonium formate (Sigma) in HPLC grade methanol (Sigma). All enzyme assays were technically and experimentally duplicated. FRET based assay^{36 (link)} was performed using 20 μM substrate ES.173.cds ^{36 (link)}, 50 ng nCDase, 75 mM NaCl, 15 mM sodium phosphate (pH 7.4), 0.3% Triton X-100 for 3 hours in 96 black well plates. Excitation wavelength was 347 nm, emission was measured at 430 nm and 530 nm and the 430 nm/ 530 nm ratio generated.