Example of Z′ score calculation for TR-F of IAEDANS-labeled F-actin binding to unphosphorylated and phosphorylated C0–C2.(A) Fluorescence waveform of IAEDANS-labeled F-actin (blue line) and the same together with 20 µM C0–C2 (black line; both normalized to their maximum intensity values) and the instrument response function (IRF; gray line). Red box (inset) highlights Relative Fluorescence Intensity ∼1/e magnified to show difference in TR-F lifetimes of actin and actin + C0–C2. This is expanded further in B. (B) Magnified view of lifetime differences from red box in A. The actual lifetimes (∼17.5 ns, shown in C–E) are the times shown on the x axis to reach 1/e of the peak intensity (∼22.5 ns) minus the time to reach peak intensity (∼5 ns in the convoluted fluorescence waveform; see peak in A). (C) Lifetimes measured in a 384-well plate containing 60 wells each of 1 µM IAEDANS–actin alone (blue), actin plus 2.5 µM C0–C2 (black), or actin plus 2.5 µM PKA-treated C0–C2 (red). (D) Comparing actin alone to actin plus C0–C2. (E) Comparing actin plus C0–C2 versus actin plus PKA-treated C0–C2. For D and E, horizontal solid lines indicate 3× SD of the mean lifetime (dotted line). Z′ score is defined as the difference between 3× SD (a) divided by the difference in the mean signal (b) in D and E. While comparisons made in D and E, having no overlap at 3× SD, are clearly significantly different, note that even the difference between actin alone and actin bound to phosphorylated C0–C2 (blue versus red in C) is also significant (P < 0.0001).