We use paramagnetic colloidal particles (Dynabeads M-270, Invitrogen) with diameter d = 2.8 μm, density ρ = 1.3 g cm−3, and magnetic volume susceptibility χ = 0.4. The particles are coated with surface carboxylic acid group with an active chemical functionality of 150 μmol × g−1 per particle. When dispersed in highly deionized water (18.2 MΩ × cm−1, MilliQ system), hydrogen ions (H+) dissociate from such groups, leaving a negative charged surface and inducing the formation of a double layer. The solution containing the particles is introduced by capillarity in a rectangular microtube made of borosilicate glass (inner dimensions 0.1 × 2.0 mm; CMC Scientific) that is immediately sealed. The particles sediment close to a glass plate, where they remain quasi-2D confined due to gravity, displaying a small thermal motion. The sample is placed in the center of a triaxial coil system arranged on the stage of a light microscope (Eclipse Ni, Nikon). External time-dependent magnetic fields are generated by passing an alternate current through the coils via a waveform generator (TGA1244, TTi) connected to different power amplifiers (AMP-1800, AKIYAMA, and BOP 20-10M, Kepco). The particle position and dynamics are extracted using digital video-microscopy with a charge-coupled device camera (Scout scA640-74fc, Basler) working at 50 frames per second.
Dynabeads m 270
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dynabeads M-270 are superparamagnetic beads that can be used for a variety of laboratory applications. They have a uniform size distribution and a high magnetic responsiveness, making them suitable for magnetic separation and capture of target molecules or cells.
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61 protocols using dynabeads m 270
1
Magnetic Particle Dynamics in Microtubes
2
Glycerol-Suspended Magnetic Bead Characterization
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Superparamagnetic beads with diameter of 2.8 μm were obtained from Dynabeads M270, Invitrogen, USA. Analytical reagent grade glycerol 99+% was from Fisher Scientific, UK. Glycerol was either used as 99% stock or 50% dilution with DI water for mixing with M270 magnetic beads. For a few hours before experiment, glycerol solution was incubated on benchtop to allow large trapped air-bubbles to escape, and magnetic beads were introduced to glycerol and mixed gently to prevent air-bubbles formation. Suspension had final concentration of 600 to 700 /μl magnetic beads, equivalent to 6-7 beads per 107 μm3 of suspension volume. Beads concentration was kept low to minimize flow pattern and magnetic interactions from nearby particles to the particle being tracked in the camera view. In the glass tube, only particles moving in the central region (at least 100 μm distance from the upper and lower walls) were selected to be tracked in the focal plane such that the drag coefficient was not influenced by nearby walls.
3
Affinity Purification of Protein A-Tagged Bait Proteins
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Affinity purification of Protein A-tagged bait proteins was carried out as described in (23 (link)). Frozen cell grindate was rapidly thawed into TBT150 buffer with 1 mM Dithiothreitol (DTT). The resulting lysate was vortexed for 1 min, polytroned for 30 s and then cleared by centrifugation at 2600 g for 5 min at 4°C. Magnetic beads (Dynabeads M-270, Invitrogen), conjugated with rabbit IgG Ab (Sigma), were washed three times with TBT150 + DTT and added to the cleared cell lysate at a concentration of 3.75 mg (25 μl slurry)/0.5 g of cell grindate. The samples were rotated for 30 min at 4°C. After binding, the beads were magnetically harvested and then quickly washed three times with 10ml of TBT150 + DTT, and finally once with 10ml of LWB while vortexing at very low speed for 5 min.
4
Yeast Pif1 and Sub1 Interaction Assay
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Yeast Pif1 and Sub1 were overexpressed and purified as described previously (27 (link),41 (link)). Purified recombinant Sub1 or BSA were covalently cross-linked onto epoxy-activated Dynabeads M-270 (Invitrogen) according to the manufacturer's instructions. Briefly, saturating amounts of protein were used for coating 5 mg of Dynabeads M-270 at 37°C for 24 h in the presence of 1.5 M ammonium sulfate. Co-precipitation experiments were performed by incubating purified Pif1 (50 μg) with Sub1- or BSA-coated dynabeads in a 200 μl binding buffer (25 mM HEPES pH 7.4, and 100 mM NaCl) with rotation at 4°C overnight. Dynabeads were captured using a magnet and washed five times in 1 ml of buffer containing 0.2% Tween 20. Pif1 protein that co-precipitated with the beads was eluted by addition of 30 μl of Laemmli sample buffer and heating at 95°C for 10 min. The sample was resolved by 10% SDS-PAGE gel and the proteins were visualized by Coomassie staining.
5
Depletion of Mouse Plasma HNP
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HNP were depleted from mouse plasma using anti-HNP conjugated magnetic beads. Briefly, anti-HNP monoclonal antibody (100 μg; ab122884, Abcam) was mixed with Dynabeads® M-270 (3.3 × 108 beads; Invitrogen) in presence of 1 M ammonium sulfate and incubated overnight at room temperature. The coated beads were washed and resuspended at 2 × 109 beads/ml and added to 50 μl of mouse plasma. The sample was incubated with rotation overnight at room temperature. Finally, the beads were removed and the depletion was confirmed by dot blot analysis.
6
Superparamagnetic Bead Functionalization
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Superparamagnetic Dynabeads M270, 2.8 μm diameter, were used for magnetic microrheology (Invitrogen). The microspheres with streptavidin surface chemistry were passivated with a combination of PEG-biotin (MW 5 K) to prevent non-specific binding and with Atto 550-biotin (Sigma) for visualization in a ratio of 75:25, respectively. Beads were rinsed with PBS and incubated for 30 minutes in excess and then washed several times to remove the reaction mixture. Beads were vortexed each time before use.
7
DDR1 Ligand Binding Dynamics
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To couple protein/antibodies to magnetic beads, the Dynabeads Antibody Coupling Kit (14311D; Invitrogen) was used. 50ug of protein was coupled to 5 mg of Dynabeads M-270 (2.8 μm, Invitrogen Norway). HUVECs infected with DDR1-EGFP virus were incubated with the beads (coated with DDR1 ligand or antibody) prior to force application for 30 min at 37 °C. An empty glass bottom dish was covered with polystyrene beads, which served as reference beads for drift correction in each experiment. Image of DDR1-EGFP droplets was acquired with total internal reflection fluorescence microscope (TIRF, Olympus).
8
Magnetic Tweezers Protein Immobilization
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Magnetic tweezers measurements were performed in fluid chambers that were functionalized by (3-aminopropyl)-trimethoxysilane in ethanol (1% v/v, Sigma-Aldrich, St. Louis, MO, USA), and glutaraldehyde (1% v/v, Sigma Aldrich) as described by Popa et al. [17 (link)]. HaloTag amine (O4) ligand (Promega, Madison, WI, USA) was added to create an attachment point for the protein of interest. Halo-(I83)6 protein was diluted in 20 mM HEPES buffer and added to a functionalized flow cell. Chambers were washed with TRIS buffer and then streptavidin coated paramagnetic beads (Dynabeads M-270, Invitrogen, Waltham, MA, USA) were added to attach the biotinylated C-terminus of the protein.
9
Magnetic Cargo Transport with Colloidal Particles
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As magnetic colloidal particles, we use monodisperse paramagnetic microspheres from Invitrogen (Dynabeads M270), composed of a highly cross-linked polystyrene matrix and evenly doped with nanoscale superparamagnetic grains IJFe 2 O 3 and Fe 3 O 4 ). These particles are characterized by a radius a = 1.4 μm, a density ρ = 1.6 g cm -3 and a magnetic volume susceptibility χ ≈ 0.4. 26 As non-magnetic cargos, we use monodisperse microparticles based on silicon dioxide and having sizes ranging from 1 to 5 μm, which were purchased from Sigma Aldrich. These particles are diluted in deionized water and mixed at a proper concentration with the paramagnetic colloidal particles before being deposited above the FGF.
10
Characterization of Superparamagnetic Polystyrene Spheres
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We use aqueous suspensions of superparamagnetic polystyrene spheres (carboxylic acid functionalised Dynabeads ® M-270, Invitrogen), with a diameter of 3.0 µm and a mass density of ρ = 1.6 g cm -3 . Due to the magnetic nanoparticles embedded in the polymer matrix, these particles are magnetic and the magnitude of the magnetic moment is proportional to the external field for sufficiently small fields. Using SQUID (superconducting quantum interference device) measurements, we confirmed that our experiments are conducted within this region of reversible linear magnetisation and find a dimensionless volume susceptibility of χ = 0.76.
The particles are contained in a 200 µm thick quartz glass cell (Hellma) and the packing fraction φ is varied between 0.01 and 0.05. Due to their relatively high mass density, they form a mono-layer at the bottom of the sample cell. The out-of-plane fluctuations are negligible as can be inferred from the particle's gravitational height,
with ∆ρ the mass density difference between the solvent and the particle, and g the acceleration due to gravity. For our system, h g = 0.06 µm, which is only a fraction of the particle radius. The Brownian time, which is the time it takes for a particle to diffuse over a distance equal to its diameter, is given by
and is ∼20 s when these particles are suspended in a 20% ethanol solution.
The particles are contained in a 200 µm thick quartz glass cell (Hellma) and the packing fraction φ is varied between 0.01 and 0.05. Due to their relatively high mass density, they form a mono-layer at the bottom of the sample cell. The out-of-plane fluctuations are negligible as can be inferred from the particle's gravitational height,
with ∆ρ the mass density difference between the solvent and the particle, and g the acceleration due to gravity. For our system, h g = 0.06 µm, which is only a fraction of the particle radius. The Brownian time, which is the time it takes for a particle to diffuse over a distance equal to its diameter, is given by
and is ∼20 s when these particles are suspended in a 20% ethanol solution.
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