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. 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 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 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 reducing or oxidizing agent and the effect on 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.

Additional Resources:

• Photobleaching in TRIC experiments