Preparation of samples for single molecule fluorescence spectroscopy

Preparation of samples for single molecule fluorescence spectroscopy 

In this chapter we will briefly discuss the photophysical properties of commonly used dyes and, by reference to biomolecular systems studied by single molecule FRET in the literature, discuss the properties of commonly used dye pairs. We will then describe, in detail, the labelling and purification methods used in our laboratories to generate milligram quantities of a single chain protein labelled with two different dyes. Finally, we will discuss a variety of methods by which biomolecular systems have been tethered at or close to a surface, a necessary prerequisite for performing many of the experiments described in other chapters. 

---