Brightness experiments

Abstract

This chapter presents an overview of quantitative fluorescence brightness experiments with special emphasis on single-color measurements of protein homo-interactions inside living cells. We discuss practical considerations in the choice of the fluorescent labels and the calibration measurements necessary for quantitative interpretation of brightness experiments. Methods to identify and avoid common pitfalls, such as bleaching and saturation, are addressed. We examine the interpretation of brightness data with moment analysis. In particular, we focus on how to avoid or correct for undersampling, as well as how to characterize and adjust for photon detector effects. We conclude by describing brightness titration experiments which determine the binding curve and stoichiometry of a protein from apparent brightness data.