Sedimentation velocity analytical ultracentrifugation (SV-AUC) is a classic biophysical technique to determine the size and shape of macromolecules in solution, and to characterize their interactions with regard to the number, size, stoichiometry, and affinity of complexes formed in self-association and hetero-association processes. A recently introduced fluorescence detection system (FDS) extends the detection limit of SV-AUC by several orders of magnitude, permitting, for example, the study of binding events with picomolar dissociation constants. Further, the selectivity of fluorescence detection offers possibilities to apply SV to crowded solution such as biological fluids. The FDS system consists of an optics box, a switch box, a system box, and operating software. Data acquisition is controlled by several parameters including detector voltage and gain, focal depth of excitation beam, sector angle, and magnet angle. In this movie, we demonstrate how to mount the FDS detector prior to an SV experiment, and how to adjust the required control parameters in the system software to initiate fluorescence data acquisition. Finally, we present FDS specific data pre-processing steps available in the SV data analysis software SEDFIT.
