10.1371/journal.pone.0035962.g005 Jenni Leppiniemi Jenni Leppiniemi Toni Grönroos Toni Grönroos Juha A. E. Määttä Juha A. E. Määttä Mark S. Johnson Mark S. Johnson Markku S. Kulomaa Markku S. Kulomaa Vesa P. Hytönen Vesa P. Hytönen Tomi T. Airenne Tomi T. Airenne ITC analysis of ligand binding. Public Library of Science 2012 ligand 2012-05-04 00:54:35 Figure https://plos.figshare.com/articles/figure/_ITC_analysis_of_ligand_binding_/313275 <p>Thermograms of measurements performed at three different temperatures (<b>A</b>) 15°C, (<b>B</b>) 25°C and (<b>C</b>) 40°C are shown. At each temperature, core-bradavidin was first titrated with Brad-tag (1), followed by competitive titration with biotin (2). As a control measurement, core-bradavidin was titrated with biotin only (3). In order to prove that the intrinsic Brad-tag decreases the affinity towards biotin, wt bradavidin was also titrated with biotin (4). In addition, core-bradavidin was titrated with Brad-tag–EGFP at 15 and 25°C (5). (<b>D</b>) Comparison of the binding enthalpies of all measurements at different temperatures. Brad-tag had a clear effect on the binding enthalpy of the competitive titration with biotin at 15°C (endothermic Brad-tag binding) and 40°C (exothermic Brad-tag binding). At 25°C, the enthalpy of competitive titration was equal to that of titration with biotin only (no detectable binding of Brad-tag to core-bradavidin).</p>