ATP boosts protein solubility: Adenosine triphosphate (ATP) has well-characterized roles in providing energy for biochemical reactions within cells. Patel et al. find that ATP may also enhance protein solubility, which could help explain why such high concentrations of ATP are maintained in cells (see the Perspective by Rice and Rosen). Protein concentrations in cells can exceed 100 mg/ml. The authors found that ATP at concentrations found in cells could act as a hydrotrope to help solubilize hydrophobic proteins. The results raise the possibility that ATP concentrations could influence processes such as protein aggregation that occur in disease or liquid-liquid phase separations that occur within cells. Science , this issue p. 753 ; see also p. 701

Rheology of aging protein condensates: Protein condensates that form by undergoing liquid-liquid phase separation will show changes in their rheological properties with time, a process known as aging. Jawerth et al. used laser tweezer–based active and microbead-based passive rheology to characterize the time-dependent material properties of protein condensates (see the Perspective by Zhang). They found that condensate aging is not gelation of the condensates, but rather a changing viscoelastic Maxwell liquid with a viscosity that strongly increases with age, whereas the elastic modulus stays the same. Science , this issue p. 1317 ; see also p. 1271

The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high-quality proteins, standardized procedures, and generally agreed-upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins.

An increasing number of proteins with intrinsically disordered domains have been shown to phase separate in buffer to form liquidlike phases. These protein condensates serve as simple models for the investigation of the more complex membraneless organelles in cells. To understand the function of such proteins in cells, the material properties of the condensates they form are important. However, these material properties are not well understood. Here, we develop a novel method based on optical traps to study the frequency-dependent rheology and the surface tension of P-granule protein PGL-3 condensates as a function of salt concentration. We find that PGL-3 droplets are predominantly viscous but also exhibit elastic properties. As the salt concentration is reduced, their elastic modulus, viscosity, and surface tension increase. Our findings show that salt concentration has a strong influence on the rheology and dynamics of protein condensates suggesting an important role of electrostatic interactions for their material properties.