| Assay Method Information | |
| | Assay of FatA Acyl-ACP Thioesterase |
| Description: | To determine the level of FatA thioesterase activity in the presence of test compounds, an assay buffer consisting of 50 mM Tris-HCl pH 8.0, 0.15M NaCl, 5 mM EDTA, 10 μM oleoyl-CoA is combined with enzyme (1-80 nM Arabidopsis FatA thioesterase) to a final volume of 100 μl. The assay is carried out in black 96-well microtitre plates into which test compounds dissolved in DMSO (1% v/v final assay concentration) have been dispensed prior to the addition of other assay components. Once all assay components have been added, the plates are incubated at room temperature for 5 min during which time the reaction progresses at a linear rate. Enzymatic activity is simultaneously stopped, and detection reagent added, by the addition of 10 μl solution of 220 μM 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (“CPM”) dissolved in 50% v/v ethanol. After an appropriate time to allow for near complete reaction of the CoASH formed by the enzyme with CPM, the fluorescence is read in a plate reader using excitation and emission wavelengths of 390 nm and 470 nm respectively. The assay signal associated with maximal (uninhibited) enzyme activity is determined with DMSO present in the reaction at 1% v/v final concentration and the assay signal associated with full enzyme inhibition is determined from wells to which no enzyme has been added. |
| Affinity data for this assay | |
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