FIG 2.

Optimization of the SPA competition assay. (A) Detergent optimization of S/B. Shown are the results from Brij 58 titration in reaction mixtures containing 1.25 mg/ml beads, 0.125 μM bhCA-1, and 0.125 μM [³H]PF74 (assay signal) or the equivalent lacking bhCA (background). (B) Optimization of [³H]PF74 concentration. Two biotinylated CA hexamer preparations (bhCA-1 and bhCA-2) were tested at 0.125 μM concentration in reaction mixtures containing 1.25 mg/ml beads and 0.2% (wt/vol) Brij 58. (C) Optimization of the concentrations of the SPA beads. (D) Optimization of bhCA-2 concentration. Reaction mixtures in panels C and D contained 0.2% (wt/vol) Brij 58 and 0.1 μM [³H]PF74. (E) Specificity of the assay signal and stability of the assay with time. Background was established with assay mixture lacking bhCA (no bhCA). 5Mut–bhCA-1, which exhibits low affinity for PF74, was prepared by direct biotinylation of the 5Mut CA hexamer.