Measuring Molecular Diffusion in Self-Organizing Xenopus Extracts by Fluorescence Correlation Spectroscopy

The cytoplasm is densely packed with macromolecules and organelles, displaying viscoelastic properties at various scales. How biochemical reactions function efficiently enough in a seemingly jammed environment remains elusive. Cell-free Xenopus laevis extracts represent a powerful system for investigating the biochemistry and biophysics of living systems. Here we present a protocol for characterizing macromolecular diffusion in self-organizing cytoplasmic extracts using fluorescence correlation spectroscopy (FCS), which measures the motions on a distance scale of ~200 nm. The method can also be used to characterize diffusion in the cytoplasm as it progresses through different phases of the cell cycle.