Visualization and Quantitation of Phase-Separated Droplet Formation by Human HP1α

Methods Enzymol. 2018:611:51-66. doi: 10.1016/bs.mie.2018.09.034. Epub 2018 Oct 31.

Abstract

The ability of the heterochromatin protein-1 (HP1) to phase separate into droplets suggests new mechanisms of gene organization in the cell nucleus. An accumulating body of work suggests that other nuclear proteins also display phase separation behaviors in vitro. To understand the mechanistic and biological significance of such droplet formation a rigorous biophysical characterization of this behavior is necessary. Herein we describe procedures for imaging HP1 droplets by brightfield microscopy, and two methods to quantify phase separation.

Keywords: Chromatin; HP1; Phase separation.

MeSH terms

  • Buffers
  • Cell Culture Techniques / methods
  • Cell Nucleus / chemistry
  • Chromatography, Gel / methods
  • Chromobox Protein Homolog 5
  • Chromosomal Proteins, Non-Histone / chemistry*
  • Equipment Design
  • Humans
  • Microscopy / instrumentation
  • Microscopy / methods*
  • Nephelometry and Turbidimetry / methods
  • Phase Transition*
  • Spectrophotometry / instrumentation
  • Spectrophotometry / methods*

Substances

  • Buffers
  • CBX5 protein, human
  • Chromosomal Proteins, Non-Histone
  • Chromobox Protein Homolog 5