Steady-state binding kinetics
Experiments that measure the proportion of target proteins occupied by a ligand when the ligand has reached a state of equilibrium binding with the population of target proteins are referred to as steady-state binding kinetic experiments. Binding parameters such as KD and Hill slope are obtained from plots of steady-state binding data. Generally, when people refer to a degree of binding to a target observed at a particular concentration of a ligand, they are referring to steady-state binding at equilibrium. In contrast, transient-state kinetic data are obtained during the (often brief) period between addition of a ligand to a population of target proteins and a state of equilibrium binding being achieved.
A steady-state binding curve should contain several data points that show the proportion of targets occupied at several different ligand concentrations. Though published binding curves often contain as few as 5 points (obtained at 5 concentrations of ligand), more points are always better, and ideally no fewer than 6 points spanning a concentration range between 0.5 × KD and 5 × KD should be considered adequate. In the author’s laboratory, binding curves always contain at least 10 data points spanning a concentration range between 0.3 × KD and 8 × KD.
If there is an opportunity to make replicate measurements of binding at the chosen ligand concentrations, these should not be made at the expense of reducing the number of distinct ligand concentrations that can be assayed. For example, if an experimental assay set-up allowed for 12 individual measurements to be made in one assay, it is preferable to include single measurements of binding made at 12 distinct ligand concentrations, rather than duplicate measurements made at 6 distinct ligand concentrations.
It is necessary when designing an experiment to have some idea of the KD value beforehand, so that appropriate concentrations of ligand may be prepared. If the KD is unknown, a range-finding experiment should be done first; this involves making single measurements of binding at concentration intervals roughly 0.5 log units apart, over a wide range within which there is some confidence that the KD lies (for example, 0.1 nM to 100 nM). With an estimate of KD in hand, concentrations should be chosen
