PptT SPA assay for high throughput screening.
A. Principle of the PptT SPA assay. B. Effect of enzyme concentration on the SPA assay signal. Biotinylated apo-ACPb (4 µM) and radiolabeled [3H]CoA (2 µM) were incubated in the presence of 0, 40, 80, or 160 nM of PptT in standard conditions (see materials and methods). Reactions were stopped after various times by addition of stop buffer and 250 µg of SPA beads resuspended in 60 µl of water. Signals were detected by scintillation counting using a TopCount (Perkin Elmer). A parallel kinetic experiment was carried out with 80 nM of enzyme: the reaction was stopped at various times and loaded onto a urea polyacrylamide gel to separate apo- (a) and holo-ACP (h) forms (right panel). The gel was stained with Coomassie blue. C. Determination of the Z′ factor. SPA assays were carried out in standard conditions (4 µM of ACPb, 2 µM of [3H]CoA, 2 µM of CoA) in the presence of 80 nM of enzyme (n = 20, black squares) or without enzyme (n = 20, black circles) at 30°C for 1.5 hour in a 96-well plate. Reactions were stopped by addition of stop buffer and SPA beads, and scintillation signals were detected using a TopCount. The Z′ factor was calculated as indicated in materials and methods (μc+ = 100, μc− = 5.53, σc+ = 4.74, σc− = 0.42). The dotted lines correspond to three standard deviations from the mean of the positive and negative controls. Data are expressed relative to the mean value for positive controls.