| Assay Method Information | |
| | Enzymatic Assay |
| Description: | r-AC protein samples were pre-incubated with various concentrations of test compounds or vehicle control in 100 mM NaH2PO4/citrate buffer pH 4.5, 0.1% Nonidet P-40, 3 mM DTT for 30 min at 37° C. Samples were incubated with 100 μM N-lauroyl ceramide (Nu-Chek Prep, Elysian, Minn.) at 37° for 30 min. The reaction was stopped by addition of a mixture of chloroform/methanol (2:1 vol/vol) containing 1 nmol of heptadecanoic acid (HDA; NuChek Prep). The organic phases were collected, dried under nitrogen, and analyzed by LC/MS in the negative-ion mode using heptadecanoic acid (HDA) as internal standard (m/z=199 for lauric acid, m/z=269 for HDA). HDA was eluted on an XDB Eclipse C18 column isocratically at 2.2 mL/min for 1 min with a solvent mixture of 95% methanol and 5% water, both containing 0.25% acetic acid, and 5 mM ammonium acetate. The column temperature was 50° C. |
| Affinity data for this assay | |
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