| Assay Method Information | |
| | Assay of ABAD Enzymatic Activity |
| Description: | The assay for the inhibition of reduction of S-acetoacetyl-CoA (SAAC) by ABAD was carried out with ABAD (418 ng/ml), SAAC (172 μM), NADH (102 μM), and different concentrations of inhibitors (from 0 to 1000 μM) in 93 mM potassium phosphate buffer (pH 7.3). Before the assay, all the assay components except SAAC were pre-incubated for 5 minutes, and the reaction started with the addition of SAAC into the reaction mix. The reaction was carried out for a total 6 minutes at room temperature under steady-state conditions, and the decrease of NADH absorbance at 340 nm was determined every 10 seconds. Kinetic data were analyzed by PRISM (Scitech, San Diego, Calif.) to determine IC50 values and Ki. One unit of enzyme activity was defined as that which converted 1.0 μmol of substrate to product per minute. |
| Affinity data for this assay | |
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