Abstract

Biomolecular condensates formed via liquid–liquid phase separation (LLPS) are dynamic compartments critical for spatiotemporal regulation of key cellular processes, including RNA metabolism. However, under genetic mutations or chronic cellular stress, these condensates can transition into less dynamic, gel- or solid-like structures—often associated with neurodegenerative disorders.

This webinar will cover a comprehensive experimental workflow to characterize the material properties and molecular dynamics of protein condensates. Topics include the expression and purification of full-length recombinant mouse prion protein (PrP) in E. coli, serving as an in vitro model system. The session will also highlight optimized protocols for fluorescence recovery after photobleaching (FRAP) in both in vitro and cellular settings, along with strategies for combining differential interference contrast (DIC) and fluorescence microscopy to visualize condensate morphology and protein folding states. In addition, the application of X-ray photon correlation spectroscopy (XPCS) will be discussed as a powerful technique to assess nanoscale dynamics and condensate fluidity.

Together, these complementary techniques offer robust insights into the folding states, structural transitions, and physical behavior of proteins within condensates—providing valuable tools to better understand condensate dysregulation in disease.

Highlights

- FRAP protocols for analyzing condensate dynamics in vitro and in cells using spinning disk confocal microscopy.

- Combined DIC/fluorescence imaging for detailed visualization of condensate morphology.

- Introduction to XPCS for probing nanoscale biomolecular motion and condensate fluidity.

- Discussion on phase transitions of condensates and their relevance to neurodegenerative disease.