Figure 3

Anisotropy titration assay. A) Typical direct titration curve of a solution of 1.25 μ M Xlp53 titrated into 20 nM labelled Ref_Alexa488. Measured anisotropy values were fitted to a Hill equation allowing the calculation of the dissociation constant K_d for the binding event between the labelled DNA and the protein. B) Displacement of a labelled reporter oligonucleotide from the complex by an unlabelled competitor oligonucleotide, reflected by a decrease in the anisotropy, allows accurate measurement of the difference in the K_d between two sequences. Shown are typical titration curves for a competition experiment, in this case Xlp53: a 50 μM solution of competitor DNA was titrated into a solution of 20 nM labelled DNA and 400 nM Xlp53. Measured anisotropies are shown for a tight (Ref, logK_d = -7.08, squares, straight line), an average (A5G, logK_d = -6.64, circles, dashed line) and a weak (G7C, logK_d = -5.70, triangles, dotted line) binding sequence with corresponding fits.