Ultracentrifugation was used for two reasons: comparison study with newExoChip and DiO-stained EV preparation. EVs were isolated using two different ultracentrifuge, Sorvall ultracentrifuges (ThermoFisher, USA) and Airfuge ultracentrifuge (Beckman Coulter, USA), depending on the sample volume. For the comparison study of model samples, the initial volume was 200μl and we used the Airfuge ultracentrifugation using an A-100/40 angle rotor for 30 minutes at 100,000xg. After the first centrifugation, 170μl of supernatant was removed from the tube and replaced with 170μl of pre-filtered PBS, and then followed by another same centrifugation step. For the comparison study of clinical samples, the same volume of initial plasma sample was used but diluted into PBS buffer. After initial ultracentrifugation at 100,000xg for 90 minutes, we aspirated the supernatant and injected another 38ml of PBS for 2nd centrifugation at 100,000 g for 90 minutes. The pellet after the 2nd centrifugation was gently spiked into 100μl of PBS and compared to the resultant from newExoChip. For the preparation of DiO stained EV, we used the same rpm conditions but performed an additional centrifugation to remove excess dye debris.
Airfuge ultracentrifuge
Manufactured by Beckman Coulter
Sourced in United States, Canada
The Airfuge Ultracentrifuge is a laboratory equipment designed for rapid and efficient separation of particles and molecules in a sample. It utilizes high-speed centrifugation to create a strong gravitational force, enabling the separation of components based on their differences in size, density, and sedimentation rate. The Airfuge Ultracentrifuge is a compact and versatile instrument that can be used in various applications within the field of biochemistry, molecular biology, and cell biology.
Automatically generated - may contain errors
Lab products found in correlation
R axis htc imaging plate detector, by Rigaku (1 mentions)
Varimax hr confocal optics, by Rigaku (1 mentions)
Taxol, by Merck Group (1 mentions)
Polyallomer centrifuge tube, by Beckman Coulter (1 mentions)
Anti rabbit igg conjugated with peroxidase, by Merck Group (1 mentions)
Ecl prime western blotting detection reagent, by Cytiva (1 mentions)
P8340, by Merck Group (1 mentions)
7 protocols using airfuge ultracentrifuge
1
Ultracentrifugation for EV Isolation and Analysis
2
X-ray Diffraction Analysis of Amyloid Fibrils
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Fibrils were spun down and washed with water three times to remove any salt. Fibrils of spine segments were spun down using a tabletop microfuge. Full-length hIAPP fibrils and spine segment seeds were spun down using an Airfuge Ultracentrifuge set at 75,000 rpm for 1 hr (Beckman-Coulter, Brea, CA). The samples were concentrated 10x in water and applied between two capillary ends and then the samples were left to dry overnight. Dried fibrils of spine segments and full-length hIAPP in Figure 3D were analyzed with a RIGAKU R-AXIS HTC imaging plate detector using Cu K(alpha) radiation from a FRE+ rotating anode generator with VARIMAX HR confocal optics (Rigaku, Tokyo, Japan). Fiber diffraction from full-length hIAPP fibrils used in Figure 6 was recorded by an ADSC Q315 CCD detector at the Advanced Photon Source 24-ID-E beamline (Argonne, IL).
Radial profiles were calculated using a program written in-house. The program calculates the average intensity as a function of distance from the beam center.
Radial profiles were calculated using a program written in-house. The program calculates the average intensity as a function of distance from the beam center.
3
Preparation of Rhodamine-Labelled Microtubules
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Single-cycled, rhodamine-labelled, GMPCPP-stabilized microtubules were prepared by incubating 2 µM tubulin containing 25% rhodamine-labelled tubulin in BRB80 (80 mM PIPES pH 6.9, 1 mM MgCl2, 1 mM EGTA) supplemented with 1 mM GMPCPP for 2 h at 37°C. Microtubules were pelleted using a Beckman Airfuge Ultracentrifuge and resuspended in BRB80 buffer to be used directly.
Double-cycled, GMPCPP-stabilized microtubules were made as described in [29 (link)]. These microtubules were stored in liquid N2 and thawed at 37°C immediately prior to use.
The concentration stated for microtubules is the concentration of polymerized tubulin.
Double-cycled, GMPCPP-stabilized microtubules were made as described in [29 (link)]. These microtubules were stored in liquid N2 and thawed at 37°C immediately prior to use.
The concentration stated for microtubules is the concentration of polymerized tubulin.
4
Ultracentrifugation of Biological Samples
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The samples of variation A, D and F were then ultracentrifuged using a Beckman Coulter Airfuge Ultracentrifuge at 178,000 g for 1 hour (30 psi for 1 hour) at room temperature. The supernatant was discarded, and the pellet was suspended in 130 µL of sterile 1XPBS.
5
Isolation of Microtubules from Fignl1-Expressing Cells
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COS-7 cells transfected with pCS2+-HA or pCS2+-HA-Fignl1 were collected in 120 µl MEM buffer (0.1 M 2-morpholinoethane sulfonic acid, 1 mM EGTA, and 0.5 mM MgCl2, pH 6.9) supplemented with a cocktail of protease inhibitors (Roche) and sonicated (total lysate). Homogenates were spun at 150,000 g for 10 min at 4°C in an Airfuge ultracentrifuge (Beckman Coulter). The supernatant was collected and supplemented with 20 µM taxol (Sigma-Aldrich) and 1 mM GTP. MTs were left to polymerize for 30 min at 37°C. The resulting extract was layered onto a 10% (wt/vol) sucrose cushion (one third of the final volume) in MEM and spun at 180,000 g for 15 min at 37°C. After centrifugation, the supernatant was carefully removed, and the pellet was resuspended in MEM buffer supplemented with protease inhibitors. The different fractions were assessed for Fignl1 and α-tubulin detection by WB by using HA and α-tubulin antibodies, respectively.
6
Sucrose Gradient Fractionation of Proteoliposomes
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50 μl of proteoliposomes in 40% (w/v) sucrose were put into the bottom of polyallomer centrifuge tubes (Beckman Coulter) and sequentially overlaid with 100 μl of 20% sucrose solution and 50 μl of 5% sucrose solution, forming a discontinuous gradient from bottom to top. The samples were centrifuged at 100,000 g for 60 min at 4°C in an Airfuge Ultracentrifuge (Beckman Coulter) using the A-100/18 rotor. Fractions of 40 μl each were collected from top to bottom and protein was detected by western blotting.
Samples were run in 10% or 15% polyacrylamide gel, blotted onto nitrocellulose membrane, and probed with anti-KtrB polyclonal antibody (overnight incubation at 4°C) or anti-KtrA polyclonal antibody (2 h at room temperature), respectively. Detection was done by incubation with anti-rabbit IgG conjugated with peroxidase (Sigma) for 30 min at room temperature and using Amersham ECL Prime western blotting detection reagents.
Samples were run in 10% or 15% polyacrylamide gel, blotted onto nitrocellulose membrane, and probed with anti-KtrB polyclonal antibody (overnight incubation at 4°C) or anti-KtrA polyclonal antibody (2 h at room temperature), respectively. Detection was done by incubation with anti-rabbit IgG conjugated with peroxidase (Sigma) for 30 min at room temperature and using Amersham ECL Prime western blotting detection reagents.
7
Biochemical Assay for Protein Aggregation
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A biochemical assay was used to detect insoluble aggregated proteins according to a previously described protocol, with some modifications [60] (link). Mammalian cells or C. elegans were extracted in 200 µl of buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0), 0.5% NP-40, 50 µM iodoacetamine, and protease inhibitor (P8340, Sigma-aldrich) by using a Bioruptor ultrasonicator at 4°C for 5 min. The lysates were then transferred to an Airfuge ultracentrifuge (Beckman Coulter) and centrifuged at 25 psi (∼130,000 g) for 5 min.
The supernatant was transferred to clean tubes and saved as the “soluble” fraction. The remaining pellet was again resuspended in extraction buffer, then sonicated for 5 min. This resuspended pellet was applied to the Airfuge and centrifuged at 25 psi for 5 min. The remaining pellet was transferred to 100 µl of resuspension buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0), 0.5% NP-40, 0.5% deoxycholic acid, and 2% SDS, and sonicated for 5 min. This fraction was considered the “insoluble” protein aggregate fraction.
The supernatant was transferred to clean tubes and saved as the “soluble” fraction. The remaining pellet was again resuspended in extraction buffer, then sonicated for 5 min. This resuspended pellet was applied to the Airfuge and centrifuged at 25 psi for 5 min. The remaining pellet was transferred to 100 µl of resuspension buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0), 0.5% NP-40, 0.5% deoxycholic acid, and 2% SDS, and sonicated for 5 min. This fraction was considered the “insoluble” protein aggregate fraction.
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