In some rare cases, Ligand binding can change the Photobleaching properties of the fluorescent Target. This means that the Photobleaching behavior changes with the ligand concentration (see Concentrations). Apart from binding-induced changes in photobleaching, some compounds and other substances can act as reducing or oxidizing agents, and thereby alter photobleaching in a concentration dependent manner, without actually binding the target.
To discriminate between binding-specific and unspecific effects on photobleaching, it is recommended to repeat the binding experiment with a negative control (see Control Experiment) target (a non-binding target, such as binding impaired mutants or unrelated proteins, or fluorophore only). If in such a control experiment the ligand still affects photobleaching behavior, it is acting as a reducing or oxidizing agent and the effect on the photobleaching rate is unspecific. If there is no effect on photobleaching observed in the control experiment, the effect observed in the original binding experiment was binding- specific.