Open Access
Issue
Published:
05 December 2018
Protocol for analyzing protein liquid–liquid phase separation
)
Download citation
320
Views
24
Downloads
63
Crossref
N/A
WoS
0
Scopus
1
CSCD
Numerous cellular functions occur in spatially and temporally confined regions. Recent studies have shown that membrane-less organelles and compartments in the cell are assembled via liquid–liquid phase separation (LLPS). In vitro LLPS assays using recombinant expressed and purified proteins are necessary for us to further understand how the assembly of phase-separated compartments is regulated in cells. However, uniform standards and protocols are lacking for these in vitro studies. Here, we describe a step-by-step protocol commonly used to investigate in vitro LLPS using purified proteins. This protocol includes expression and purification of the studied proteins, induction of LLPS of the purified proteins, and studies of the biophysical properties of the liquid droplets formed by LLPS. These protocols can be easily followed by researchers to investigate the LLPS behaviors of proteins of interest.
menu
Protocol for analyzing protein liquid–liquid phase separation
)
Abstract
Numerous cellular functions occur in spatially and temporally confined regions. Recent studies have shown that membrane-less organelles and compartments in the cell are assembled via liquid–liquid phase separation (LLPS). In vitro LLPS assays using recombinant expressed and purified proteins are necessary for us to further understand how the assembly of phase-separated compartments is regulated in cells. However, uniform standards and protocols are lacking for these in vitro studies. Here, we describe a step-by-step protocol commonly used to investigate in vitro LLPS using purified proteins. This protocol includes expression and purification of the studied proteins, induction of LLPS of the purified proteins, and studies of the biophysical properties of the liquid droplets formed by LLPS. These protocols can be easily followed by researchers to investigate the LLPS behaviors of proteins of interest.
References(10)
Aguzzi A, Altmeyer M, (2016) Phase separation: linking cellular compartmentalization to disease.Trends Cell Biol 26(7):547-558
Banani SF, Lee HO, Hyman AA, Rosen MK, (2017) Biomolecular condensates: organizers of cellular biochemistry.Nat Rev Mol Cell Biol 18(5):285-298
Bergeron-Sandoval LP, Safaee N, Michnick SW, (2016) Mechanisms and consequences of macromolecular phase separation.Cell 165(5):1067-1079
Carisey A, Stroud M, Tsang R, Ballestrem C, (2011) Fluorescence recovery after photobleaching.Methods Mol Biol 769:387-402
Dao TP, Kolaitis RM, Kim HJ, O’Donovan K, Martyniak B, Colicino E, Hehnly H, Taylor JP, Castaneda CA, (2018) Ubiquitin modulates liquid-liquid phase separation of UBQLN2 via disruption of multivalent interactions.Mol Cell 69(6):965-978
Nott TJ, Petsalaki E, Farber P, Jervis D, Fussner E, Plochowietz A, Craggs TD, Bazett-Jones DP, Pawson T, Forman-Kay JD, Baldwin AJ, (2015) Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.Mol Cell 57(5):936-947
Saha S, Weber CA, Nousch M, Adame-Arana O, Hoege C, Hein MY, Osborne-Nishimura E, Mahamid J, Jahnel M, Jawerth L, Pozniakovski A, Eckmann CR, Julicher F, Hyman AA, (2016) Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism.Cell 166(6):1572-1584
Shin Y, Berry J, Pannucci N, Haataja MP, Toettcher JE, Brangwynne CP, (2017) Spatiotemporal control of intracellular phase transitions using light-activated optodroplets.Cell 168(1–2):159-171
Su X, Ditlev JA, Hui E, Xing W, Banjade S, Okrut J, King DS, Taunton J, Rosen MK, Vale RD, (2016) Phase separation of signaling molecules promotes T cell receptor signal transduction.Science 352(6285):595-599
Zeng M, Shang Y, Araki Y, Guo T, Huganir RL, Zhang M, (2016) Phase transition in postsynaptic densities underlies formation of synaptic complexes and synaptic plasticity.Cell 166(5):1163-1175
Publication history
Copyright
Acknowledgements
They authors would like to thank the Strategic Priority Research Program of the Chinese Academy of Sciences (CAS) (XDB19000000), the National Natural Science Foundation of China (31561143001, 31630048, 31790403, and 31421002), the National Chinese Ministry of Science and Technology (2017YFA0503401), and the Key Research Program of Frontier Sciences, CAS (QYZDY-SSW-SMC006) for research funding.
Rights and permissions
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
FEEDBACK 
TOP