Figure 6.

FAM fluorescence data for the target association of the Egr-1 zinc-finger protein with the 113-bp probe DNA (D_tot = 2.5 nM) in the presence of 28-bp competitor DNA (C_tot = 2 μM). The buffer conditions were 10 mM Tris•HCl (pH 7.5), 80 mM KCl, and 200 nM ZnCl₂. (A) Change in the FAM emission spectra upon protein binding. The black solid line is the spectrum recorded for the probe DNA only; the green dotted line, for the probe DNA plus competitor DNA; and the magenta solid line, with the probe DNA, competitor DNA, and protein (P_tot = 100 nM). (B) FAM fluorescence intensity measured as a function of P_tot in the presence of the competitor DNA. (C) Stopped-flow time-course data of the FAM fluorescence intensity immediately after mixing the protein solution (P_tot = 50 nM) with the solution containing the probe and competitor DNA duplexes. The red curve represents the best fit to a mono-exponential function. (D) Protein-concentration dependence of the apparent pseudo-first-order kinetic rate constant k_app for target association. The apparent second-order rate constant for association was determined to be 4.5 × 10⁷ M⁻¹ s⁻¹. The error bars represent the standard deviations for 8 – 10 replicates.