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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230745	1/1	Escherichia coli	MG1655	(d)(U)C DG Filtration	Zhao H	2017	56%
Study summary Full title Isolation of bacterial compartments to track movement of protein synthesis factors. All authors Zhao H, Martinis SA Journal Methods Abstract Aminoacyl-tRNA synthetases (AARSs) comprise an enzyme family that generates and maintains pools of a (show more...)Aminoacyl-tRNA synthetases (AARSs) comprise an enzyme family that generates and maintains pools of aminoacylated tRNAs, which serve as essential substrates for protein synthesis. Many protein synthesis factors, including tRNA and AARSs also have non-canonical functions. Particularly in mammalian cells, alternate functions of AARSs have been associated with re-distribution in the cell to sites that are removed from translation. Sub-fractionation methods for E. coli were designed and optimized to carefully investigate re-localization of bacterial AARSs and tRNA that might aid in conferring alternate activities. Cell fractionation included isolation of the cytoplasm, periplasm, membrane, outer membrane vesicles, and extracellular media. Specific endogenous proteins and RNAs were probed respectively within each fraction via Western blots using antibodies and by Northern blots with primers to unique regions of the nucleic acid. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Protein markers EV: OmpA/ S3/ AlaRS non-EV: not specified Proteomics no EV density (g/ml) not specified Show all info Study aim Function Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells MG1655 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 150000 Density gradient Type Discontinuous Number of initial discontinuous layers 8 Lowest density fraction 10% Highest density fraction 50% Total gradient volume, incl. sample (mL) 3,7 Sample volume (mL) not spec Orientation Bottom-up Speed (g) 200000 Duration (min) 1200 Fraction volume (mL) 0.35 Fraction processing None Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins OmpA/ S3 Not detected EV-associated proteins AlaRS Detected contaminants not specified Not detected contaminants not specified Characterization: Lipid analysis No

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EV-TRACK ID	EV230745
species	Escherichia coli
sample type	Cell culture
cell type	MG1655
condition	Control condition
separation protocol	dUC/ Density gradient/ Filtration
Exp. nr.	1
EV-METRIC %	56