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An optogenetic proximity labeling approach to probe the composition of inducible biomolecular condensates in cultured cells.

STAR protocols (2021-08-12)
Emile Alghoul, Jihane Basbous, Angelos Constantinou
ABSTRACT

Inducible biomolecular condensates play fundamental roles in cellular responses to intracellular and environmental cues. Knowledge about their composition is crucial to understand the functions that arise specifically from the assembly of condensates. This protocol combines an optogenetic and an efficient proximity labeling approach to analyze protein modifications driven by protein condensation in cultured cells. Low endogenous biotin level ensures sharp signals. For complete details on the use and execution of this protocol, please refer to Frattini et al. (2021).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
杜氏磷酸盐缓冲盐水, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture
Millipore
Benzonase®核酸酶, ≥250 units/μL, ≥90% (SDS-PAGE), recombinant, expressed in E. coli, buffered aqueous glycerol solution
Sigma-Aldrich
杜氏改良 Eagle 培养基 - 高葡萄糖, With 4500 mg/L glucose, L-glutamine, and sodium bicarbonate, without sodium pyruvate, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
羟乙基哌嗪乙硫磺酸, ≥99.5% (titration)
Sigma-Aldrich
Triton X-100, for molecular biology
Sigma-Aldrich
水, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
生物素, ≥99% (HPLC), lyophilized powder
Sigma-Aldrich
氨苄西林 钠盐
Sigma-Aldrich
脱氧胆酸钠, ≥97% (titration)
Sigma-Aldrich
青链霉素, with 10,000 units penicillin and 10 mg streptomycin per mL in 0.9% NaCl, 0.1 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
溴化乙啶 溶液, BioReagent, for molecular biology, 10 mg/mL in H2O
Sigma-Aldrich
氯化锂, for molecular biology, ≥99%
Sigma-Aldrich
潮霉素 B 来源于吸水链霉菌, powder, BioReagent, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
乙二醇-双(2-氨基乙醚)-N,N,N′,N′-四乙酸, ≥97.0%
Sigma-Aldrich
TERGITOL 溶液, Type NP-40, 70% in H2O