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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV170028 1/1 Homo sapiens Synovial fluid DG
dUC
SEC
Foers AD 2017 100%

Study summary

Full title
All authors
Foers AD, Chatfield S, Dagley LF, Scicluna BJ, Webb AI, Cheng L, Hill AF, Wicks IP, Pang KC.
Journal
J Extracell Vesicles
Abstract
As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesic (show more...)As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesicle (EV) enrichment using standard methods. In this study of human SF, a size exclusion chromatography (SEC)-based method of EV enrichment is shown to deplete contaminants that remain after standard ultracentrifugation-based enrichment methods. Specifically, considerable levels of serum albumin, the high-density lipoprotein marker, apolipoprotein A-I, fibronectin and other extracellular proteins and debris are present in EVs prepared by differential ultracentrifugation. While the addition of a sucrose density gradient purification step improved purification quality, some contamination remained. In contrast, using a SEC-based approach, SF EVs were efficiently separated from serum albumin, apolipoprotein A-I and additional contaminating proteins that co-purified with high-speed centrifugation. Finally, using high-resolution mass spectrometry analysis, we found that residual contaminants which remain after SEC, such as fibronectin and other extracellular proteins, can be successfully depleted by proteinase K. Taken together, our results highlight the limitations of ultracentrifugation-based methods of EV isolation from complex biological fluids and suggest that SEC can be used to obtain higher purity EV samples. In this way, SEC-based methods are likely to be useful for identifying EV-enriched components and improving understanding of EV function in disease. (hide)
EV-METRIC
100% (93rd 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
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
Synovial fluid
Sample origin
Arthritis
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + SEC
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: TSG101/ Rab27b/ Annexin1/ Syntenin/ HSP70/ Flotillin-1
non-EV: Fibronectin/ Albumin/ ApoA1
Proteomics
yes
Show all info
Study aim
New methodological development, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Synovial fluid
Sample Condition
Arthritis
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Wash: time (min)
90
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
156.9
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
0.25
Highest density fraction
2
Sample volume (mL)
0.65
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 40 Ti
Speed (g)
202000
Duration (min)
1100
Fraction volume (mL)
1.05
Fraction processing
Centrifugation
Pelleting: volume per fraction
10
Pelleting: duration (min)
90
Pelleting: rotor type
Type 70.1Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
122.2
Size-exclusion chromatography
Total column volume (mL)
320
Sample volume/column (mL)
13
Resin type
Sephacryl S-500 HR
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin-1, HSP70, TSG101, Syntenin, Rab27b, Annexin1
Not detected contaminants
Albumin, ApoA1
Proteomics database
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
x
EV concentration
Yes
Particle yield
x
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
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