Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors
Fig 2
Miniaturization of the assay using phosphate standard.
(a) The effect of different volumes of standard phosphate solution was studied by adding it into 384 Perkin Elmer white proxy plates. The malachite green volume was kept at ratio of 1.25:1 (reaction volume: developing reagent volume). (b) The influence of the malachite green quantity was measured using 200pmol phosphate in 5 μL volume. (c) Z score was plotted against malachite green volume. Z score linearly decreases with malachite green volume. (d) A phosphate standard curve was generated using white plate fluorescence quenching versus absorbance assay in 96 well plates. Inset shows high sensitivity phosphate detection at lower concentrations. Our data indicate that the 5μL assay volume with 2.5μL developing reagent produces the optimum result using white proxy plates. In panels a and b, raw data was converted to OD and then % of quenching was calculated.