Binding affinity of WT and mutant EKLF for the β-globin promoter. (A) Stopped flow kinetic analyses. Increase in fluorescent emission intensity on binding of Hex-labeled double-stranded oligonucleotides composed of the β-globin EKLF binding site (50nM) to recombinant EKLF ZF domains (200nM). Each dataset is an average of 10 measurements. (B) Saturation fluorescent gel shift assays. Complex formation between recombinant WT, E325K, R331G, R328L, and R328H EKLF ZF protein (0-2500nM) with 40nM Hex-labeled double-stranded oligonucleotides composed of the β-globin EKLF binding site and surrounding residues (n = 6). Arrow indicates free Hex-labeled oligonucleotide. *Protein-DNA complexes. **The supershift band is considered nonspecific because at those protein concentrations EKLF binds both to its specific binding site (*) and to nonspecific DNA sequences. (C) Binding curve and Scatchard transformation of data from saturation fEMSAs (WT EKLF, 0-2500nM; E325K EKLF, 313-2500nM) and Hex-labeled oligonucleotide (40nM; n = 6). Quantified bands from fEMSA gels were used to construct regression curves using GraphPad Prism Version 4.03 software. [Bound] = [Bound]/[Bound] + [Free].