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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100001	1/1	Homo sapiens	Urine	(d)(U)C	Fernández-Llama P	2010	44%
Study summary Full title Tamm-Horsfall protein and urinary exosome isolation All authors Fernández-Llama P, Khositseth S, Gonzales PA, Star RA, Pisitkun T, Knepper MA Journal Kidney Int Abstract Urinary exosomes have been proposed as starting material for discovery of protein biomarkers of kidn (show more...)Urinary exosomes have been proposed as starting material for discovery of protein biomarkers of kidney disease. Current protocols for their isolation use a two-step differential centrifugation process. Due to their low density, exosomes are expected to remain in the low-speed (17,000 x g) supernatant and to sediment only when the sample is spun at high speed (200,000 x g). Analysis using western blot and electron microscopy found that urinary exosomes are also present in the low-speed pellet entrapped by polymeric Tamm-Horsfall protein, thus diminishing the procedure's reproducibility. Here we show that addition of dithiothreitol to the low-speed pellet disrupted the polymeric network, presumably by reduction of disulfide bonds linking the monomers. This modification shifted the exosomal proteins from the low- to the high-speed pellet. Also, by shifting the Tamm-Horsfall protein to the high-speed pellet, the use of dithiothreitol makes it feasible to use Tamm-Horsfall protein to normalize excretion rates of exosomal proteins in spot urines. We tested this by western blot, and found that there was a high degree of correlation between exosomal proteins and Tamm-Horsfall protein in the high-speed pellet. Since the yield of exosomes by differential centrifugation can be increased by chemical reduction, Tamm-Horsfall protein may be a suitable normalizing variable for urinary exosome studies when quantitative urine collections are not practical. (hide) EV-METRIC 44% (80th 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 Urine Sample origin NAY Focus vesicles exosomes 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 (d)(U)C Protein markers EV: Alix/ HSP70/ TSG101/ AQP2/ CD9 non-EV: Tamm-Horsfall glycoprotein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ HSP70/ TSG101/ AQP2 Detected contaminants Tamm-Horsfall glycoprotein ELISA Antibody details provided? No Detected EV-associated proteins AQP2 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

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EV-TRACK ID	EV100001
species	Homo sapiens
sample type	Urine
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	44