The Biogenesis of Endophilin B1 Containing Vesicles

Fluctuations of fluorescent light emerging from a small region of the sample were first considered more than four decades ago [1]. The technique is known as fluorescence correlation spectroscopy (FCS) and has been extended over the years to study a variety of processes. Here, we focus on a particular type of analysis that is sensitive to the stoichiometry of the complexes, namely brightness analysis. Brightness analysis and its related techniques are used to extract the concentration of the fluorescent complexes and their brightness (“the intensity of a single particle”) from the distribution of photon counts. Brightness is a unique parameter that relies on the single molecule sensitivity of FFS and provides a direct quantification of the oligomeric status of populations of GFP-tagged proteins. Since EGFP is a monomer with distinct fluorescence intensity and recombinant 2x-EGFP elicits twice the intensity, the overall brightness of a GFP-tagged protein is a direct function of its oligomeric state (Fig. 1). Thus, brightness analysis is a robust parameter for determining the stoichiometry of protein oligomerizations.