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. 2014 Dec 11;290(5):2888–2901. doi: 10.1074/jbc.M114.621789

FIGURE 4.

FIGURE 4.

Binding characteristics between c-di-AMP and PstASA. A, binding curve and Kd determination for c-di-AMP and purified His-PstASA. DRaCALAs were performed with 32P-labeled c-di-AMP and purified His-PstASA at a starting protein concentration of 300 μm, and fraction-bound and Kd values were determined from the curve as described previously (41). The average values from three independent experiments are plotted with standard deviations. B, dissociation curve and koff determination for c-di-AMP and purified His-PstASA. A 10 μm solution of purified His-PstA was incubated with 32P-labeled c-di-AMP. At time 0, 300 μm cold c-di-AMP was added, and the displacement of 32P-labeled c-di-AMP was measured at 10-, 20-, 30-, 40-, 50-, 60-, 90-, 120-, and 180-s time points. Fraction-bound values were plotted, and the koff value was determined as described previously (41). The average values from three independent experiments are plotted with standard deviations.