Buffer Gradients

Serial dilution of the ligand in assay buffer should not introduce any changes in buffer composition except the ligand concentration itself. For instance, if the ligand is dissolved in DMSO, it is important to perform the serial dilution in buffer containing the same amount of DMSO. In rare instances, the ligand titration significantly changes the experimental conditions. An appropriate counter-titration may then be useful to control for buffer effects. For example, in case the ligand is a lipid, it may help to counter-titrate a second, non- binding lipid in order to keep the overall lipid concentration constant throughout the experiment. Similarly, when investigating ion binding, try counter-titrating a similar ion to keep the ionic strength of the buffer constant. Alternatively, perform a Control Experiment with a slightly changed system like a non-binding ligand analogue.