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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
TERGITOL solution, Type NP-40, 70% in H2O
Sigma-Aldrich
Water, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Ethidium bromide solution, BioReagent, for molecular biology, 10 mg/mL in H2O
Sigma-Aldrich
HEPES, ≥99.5% (titration)
Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, ≥97.0%
Sigma-Aldrich
Lithium chloride, for molecular biology, ≥99%
Sigma-Aldrich
Dulbecco′s Phosphate Buffered Saline, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium - high glucose, With 4500 mg/L glucose, L-glutamine, and sodium bicarbonate, without sodium pyruvate, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Penicillin-Streptomycin, 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
Biotin, ≥99% (HPLC), lyophilized powder
Sigma-Aldrich
Hygromycin B from Streptomyces hygroscopicus, powder, BioReagent, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
Sodium deoxycholate, ≥97% (titration)
Millipore
Benzonase® Nuclease, ≥250 units/μL, ≥90% (SDS-PAGE), recombinant, expressed in E. coli, buffered aqueous glycerol solution
Sigma-Aldrich
Triton X-100, for molecular biology
Sigma-Aldrich
Ampicillin sodium salt