A User’s Guide for Phase Separation Assays with Purified Proteins

Abstract

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.

Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation

“…Rayleigh interference optical data were collected at 1-minute intervals for 10 hours. The velocity data were modeled with diffusion-deconvoluted sedimentation coefficient distributions c(s) in SEDFIT (https://sedfitsedphat.nibib.nih.gov/software/default.aspx), using algebraic noise decomposition and with signal-average frictional ratio and meniscus position refined with non-linear regression (38). The s-values were corrected for time and finite acceleration of the rotor was accounted for in the evaluation of Lamm equation solutions (39).…”

“…Under conditions where the LCD does not associate with the RRMs, the excluded volume of the RRMs decreases the potential for LCD-LCD mediated phase separation and the higher charge content of the RRMs has a solubilizing effect. In fact, this solubilizing property of folded domains has been used to prevent phase separation of strongly selfassociating LCDs until the solubilizing domain is proteolytically cleaved(21,22,38). The solubilizing effect of karyopherins on FUS and other RNA-binding proteins(39)(40)(41)(42) may work through similar mechanisms.The reason for the steep increase of the saturation concentration of full-length hnRNPA1 above 175 mM NaCl is not entirely clear.…”

Connecting coil-to-globule transitions to full phase diagrams for intrinsically disordered proteins

“…Intrinsically disordered proteins that have the requisite valence of adhesive linear motifsstickers -can drive phase separation and give rise to membraneless biomolecular condensates [1][2][3][4][5]. Knowledge of how phase diagrams vary with amino acid sequence and changes to solution conditions is an essential component of understanding how proteins contribute to condensate assembly and their regulated dissolution [1][2][3][4]6]. Comparative assessments of sequence-to-phase diagram relationships are also invaluable for enabling the design of novel disordered low complexity domains (LCDs) that give rise to synthetic condensates for engineering applications [7][8][9][10][11].…”

A generic approach for studying the kinetics of liquid-liquid phase separation under near-native conditions

“…Phase-separated droplets, however, are never in thermodynamic equilibrium but are in a transition towards a final state of two separate phases. Moreover, the proteins involved in these processes are aggregation prone and have a tendency to be sticky; thus, most often they are prepared either with a fused solubility tag (MBP, GFP, or GST), 7,8 or under denaturing (6-8 M urea) 9 or otherwise non-physiological (very high salt, detergents) 10 conditions, which may strongly interfere with experimental results. For example, transitions of the prion-like domain of Sup35 have been studied under two different conditions, starting from highly denaturing conditions (8M GuHCl) or from a stock of very high salt; 10 from initial denaturing conditions, it aggregated into amyloid-like fibers, 11 whereas when diluted from high salt, it phase separated into liquid droplets which later turned into gel condensates.…”

“…Due to the general biological importance of the phenomenon of LLPS, the field requires clear experimental standards for developing physiologically relevant models based on reproducible results. 10,13 Toward this goal, we propose here a generic method in which phase separation is induced by a simple pH jump at near-native conditions. To demonstrate the benefits of our approach, we compared the kinetics of LLPS of the low-complexity domain (LCD) of hnRNPA2 initiated by a pH drop (from 11.0 to 7.5) with kinetics performed in the presence of salt, urea and by cleaving its MBP solubility tag as reported in literature 9 .…”