Virus particles were concentrated with a 10–40 % sucrose gradient prepared with a Buchler gradient maker (Buchler Instruments Inc., Fort Lee, NJ, USA) with 17.5 ml of 10 % (v/v) and 17.5 ml of 40 % (v/v) sucrose in the suspension buffer (0.02 M Tris, pH 7.0, 0.001 M MgCl2). One millilitre of virus particles was overlaid on the sucrose gradient and ultracentrifuged for 2.5 h at 150000 g at 4 °C using a Beckman Coulter SW32Ti rotor. After centrifugation, fractions of 1.8 ml from top to bottom were collected and numbered from #1 to #20. Groups of three fractions were pooled, diluted in 40 ml of suspension buffer and centrifuged 2.5 h at 180000 g using a Beckman Coulter 50.2Ti rotor. The pellet was suspended in 200 µl suspension buffer. Fraction groups #10 to #12, #13 to #15 and #16 to #18 were visualized by TEM. Particles from fraction #13 to #15 were used to measure the diameter of particles with ImageJ [30 ]. The calculation of the mean, standard deviation and the mean comparison with a student t-test was performed in R.
50.2 ti rotor
Manufactured by Beckman Coulter
Sourced in United States
The 50.2 Ti rotor is a fixed-angle centrifuge rotor designed for use in Beckman Coulter ultracentrifuges. It is capable of reaching a maximum speed of 50,000 rpm and can generate a maximum relative centrifugal force (RCF) of 303,000 x g.
Automatically generated - may contain errors
Lab products found in correlation
Ufc803024, by Merck Group (4 mentions)
Sw40ti rotor, by Beckman Coulter (3 mentions)
Sephadex g 25 column, by GE Healthcare (3 mentions)
Atto647n nhs ester, by Merck Group (3 mentions)
P8112, by New England Biolabs (3 mentions)
Ni nta magnetic beads, by New England Biolabs (3 mentions)
Turbonuclease, by Abnova (2 mentions)
Ultrafiltration spin columns, by GE Healthcare (2 mentions)
200 200pro, by Tecan (2 mentions)
Clostridium perfrigens, by Merck Group (2 mentions)
Optima centrifuge, by Beckman Coulter (2 mentions)
Bac to bac baculovirus expression system, by Thermo Fisher Scientific (2 mentions)
Optima le 80k ultracentrifuge, by Beckman Coulter (2 mentions)
Pd 10 desalting column, by GE Healthcare (2 mentions)
Amicon stirred cell pressure based concentrator, by Merck Group (2 mentions)
50.4 ti rotor, by Beckman Coulter (2 mentions)
Ultracentrifuge, by Beckman Coulter (2 mentions)
Sw32ti rotor, by Beckman Coulter (1 mentions)
Nutrient broth, by Thermo Fisher Scientific (1 mentions)
Visking dialysis tubing type 8 32, by Carl Roth (1 mentions)
39 protocols using 50.2 ti rotor
1
Virus Particle Purification and Size Analysis
2
Cultivation of WT-A10 Cells Expressing HA-Tagged CagA
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WT-A10 cells, inductively expressing haemagglutinin (HA)-tagged wild-type CagA, were maintained as described previously8 (link). WT-A10 cells were cultured in RPMI1640 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS; depleted of exosomes), 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM l -glutamine, penicillin G sodium, streptomycin sulphate (1 × ), 500 μg/mL G418, 100 μg/mL hygromycin B, and 1 μg/mL doxycycline (Dox) at 37 °C in an atmosphere containing 5% CO2. Exosome-depleted FBS was prepared by ultracentrifugation at 120,000 × g for 5 h at 4 °C using a 50.2 Ti rotor (Beckman Coulter). AGS cells were cultured in RPMI1640 medium supplemented with 10% heat-inactivated FBS, 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, penicillin G sodium, streptomycin sulphate (1×), 2.2 g/L sodium bicarbonate solution, and 50 μM 2-mercaptoethanol.
3
Phage Lysate Preparation and Purification
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Standard broth lysis procedure was used to prepare phage lysates. Nutrient broth inoculated with 1% (vol/vol) cell suspension from the overnight grown culture of MAK757 and grown at 37°C for 4–6 h in a shaker incubator. Mid-logarithmic-phase cultures of V. cholerae MAK757 were infected with respective phages at a multiplicity of infection of 0.1, and the infected cultures were incubated with shaking at 37°C until complete lysis occurred. After complete lysis of the culture solution, few drops of chloroform was added in the lysate, mixed well and centrifuged at 10,000 rpm for 20 min at 4°C. Phage lysate was collected and stored at 40C followed by centrifugation. For concentrating, phage lysate was centrifuged at 30,000 rpm for 2 h at 4°C using a Beckman 50.2 Ti rotor to pellet down the phage particles, resuspended in 0.05 M Tris [pH 7.5] with 0.02 M MgCl2 and kept at 4°C.[19 (link)] Broth lysis was used for preparation of larger stock. However, to get small volume high titer stock of phages, alternate to the above lysis method, plate lysis procedure was used for phage propagation following the standard methodology.[19 (link)] Bacteriophages used in the present study were purified using cesium chloride density (1.3–1.7 g/ml) gradient as per the standard protocol.[18 (link)19 (link)]
4
Isolation and Purification of Bacterial OMVs
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The E. coli DH5α strain, E. coli K-12 BW25113 strain (WT), and E. coli deletion mutants were cultured at 37°C in LB broth. Bacteria were prepared at an OD600 of 0.0008 by Dulbecco's modified Eagle medium (DMEM) containing 4.5 g/L glucose and 0.2 μM Fe3+ (Fujifilm, Osaka, Japan) and then were cultured at 37°C for 12 h under 5% CO2 conditions. ClearColi (E. coli BL21(DE3) mutant lacking the oligosaccharide chain of LPS; Lucigen, Buenos Aires, Argentina) was gifted from Sohkichi Matsumoto (Department of Bacteriology, Niigata University Graduate School of Medical and Dental Sciences). Similarly, ClearColi bacteria were cultured at 37°C for 27 h under 5% CO2 conditions. After centrifugation at 2,000 × g for 30 min, the bacterial culture medium was collected as supernatant. After we centrifuged the bacterial culture medium at 10,000 × g at 4°C for 30 min, the supernatant was filtrated by a 0.2-μm filter (Kurabo). After we ultracentrifuged the filtrate (Ec-med) using a 50.2 Ti rotor (Beckman) at 100,000 × g at 4°C for 3 h, the supernatant (Ec-sup) was removed, and phosphate-buffered saline (PBS) was added in pellets. After we ultracentrifuged the filtrate at 100,000 × g at 4°C for 2 h, pellets (OMVs) were suspended by PBS.
5
Purification of Phage SU10 from E.coli
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Phage SU10 was propagated on E.coli strain ECOR10, grown at 37 °C in nutrient broth (Oxoid). Phage lysate from 300 ml of bacterial culture was treated with turbonuclease (Abnova) (final concentration 5 Units/ml), centrifuged at 6000 x g for 20 min at 4 °C, and filtered through a 0.45 μm polyethersulfone syringe filter. Phages from the filtrate were pelleted by centrifugation at 54,000 x g for 2.5 h at 4 °C in a 50.2 Ti rotor (Beckman Coulter). Phage pellets were dissolved by overnight incubation in 6 ml of a phage buffer (50 mM Tris-Cl, 10 mM NaCl, 10 mM CaCl2, pH 8.0) at 4 °C. Dissolved pellets were overlaid onto a step CsCl density gradient (3 ml of each CsCl solution in phage buffer with densities 1.45 g/ml, 1.50 g/ml, and 1.70 g/ml) and centrifuged at 210,000 x g for 4 h at 12 °C using an SW40Ti rotor (Beckman Coulter). Bands containing phage particles were collected using a 0.8 mm gauge needle and syringe. Caesium chloride was removed by dialysis against the phage buffer at 4 °C overnight using Visking dialysis tubing type 8/32″, 0.05 mm thick (part no. 1780.1, Carl Roth, Germany).
6
Telomerase Enzyme Purification Protocol
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The tlc1- cells (>75 generations) were grown in YPD medium at 25° overnight (ON) and harvested at OD600 = 1-1.5. The cells were resuspended in TMG buffer (10 mM Tris-HCl pH 8, 1.2 mM MgCI2, 0.1 mM EDTA, 0.1 mM EGTA, 1.5 mM DTT, 40U/ml RiboLock RNasin (Thermo Scientific), 1x Complete Protease inhibitor cocktail (Roche) and disrupted using a bead beater. The protein fraction was ultracentrifuged in a 50.2 Ti rotor (Beckman) at 40 000 rpm, 4° for 90 min. The crude extract was partially purified on a DEAE-agarose column, which was equilibrated with TMG buffer (10% glycerol) and washed with 0.4 M and 0.5 M sodium acetate in TMG buffer (10% glycerol) respectively. The active telomerase enzyme was eluted using 0.6 M sodium acetate in TMG buffer (10% glycerol). Desalting and concentrating was done using Amicon Ultra-15 centrifugal filters (Millipore, 30K MWCO). Aliquots were stored in -80°. All solutions were prepared with Diethylpyrocarbonate treated water to prevent RNase contamination.
7
Purification and Characterization of Native Mucins
8
Purification and Characterization of Native Mucins
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This study used native porcine gastric mucins (MUC5AC), porcine
intestinal mucins (MUC2), and human salivary mucins (MUC5B), which differ from
industrially purified mucins in their rheological properties and
bioactivities10 (link),46 (link). Native mucins were purified
as described previously10 (link),18 . In brief, mucus was scraped
from fresh pig stomachs and intestines and solubilized in sodium chloride.
Insoluble material was removed via ultracentrifugation at 190,000 x
g for 1 h at 4 °C (Beckman 50.2 Ti rotor with
polycarbonate bottles). Submandibular saliva was collected from human volunteers
as described above using a custom vacuum pump, pooled, centrifuged, and protease
inhibitors were added10 (link).
Mucins were purified using size-exclusion chromatography on separate Sepharose
CL-2B columns. Mucin fractions were then desalted, concentrated, and lyophilized
for storage at –80 °C. Lyophilized mucins were reconstituted by
shaking them gently at 4 °C overnight in the desired medium.
Mass spectrometry is routinely used to monitor the composition of
purified mucin extracts. This type of analysis has shown that mucin extracts
purified from porcine stomach mucus, for example, are composed predominantly of
MUC5AC, with small quantities of MUC2, MUC5B, and MUC6, as well as histones,
actin, and albumin47 (link).
intestinal mucins (MUC2), and human salivary mucins (MUC5B), which differ from
industrially purified mucins in their rheological properties and
bioactivities10 (link),46 (link). Native mucins were purified
as described previously10 (link),18 . In brief, mucus was scraped
from fresh pig stomachs and intestines and solubilized in sodium chloride.
Insoluble material was removed via ultracentrifugation at 190,000 x
g for 1 h at 4 °C (Beckman 50.2 Ti rotor with
polycarbonate bottles). Submandibular saliva was collected from human volunteers
as described above using a custom vacuum pump, pooled, centrifuged, and protease
inhibitors were added10 (link).
Mucins were purified using size-exclusion chromatography on separate Sepharose
CL-2B columns. Mucin fractions were then desalted, concentrated, and lyophilized
for storage at –80 °C. Lyophilized mucins were reconstituted by
shaking them gently at 4 °C overnight in the desired medium.
Mass spectrometry is routinely used to monitor the composition of
purified mucin extracts. This type of analysis has shown that mucin extracts
purified from porcine stomach mucus, for example, are composed predominantly of
MUC5AC, with small quantities of MUC2, MUC5B, and MUC6, as well as histones,
actin, and albumin47 (link).
9
Labeling and Purification of ZIKV Particles
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The procedure of virus labeling was performed as previously described with minor modifications (Chu et al., 2014 (link), 2019 (link)). ZIKV particles were pelleted from a ZIKV-containing supernatant by ultra-centrifugation at 47,000 rpm in a Beckman 50.2Ti rotor for 3.5 h. Virus pellets were resuspended in a HNE buffer (5 mM HEPES, 150 mM NaCl, and 0.1 mM EDTA, pH 7.4) and further concentrated by ultrafiltration spin columns (GE healthcare). Concentrated ZIKV particles were labeled with Atto647N-NHS ester (Sigma-Aldrich), with maximum absorption at 646 nm and maximum emission at 664 nm. Briefly, 1 × 107pfu/ml of ZIKV were mixed with 2 nmol of Atto 647N NHS ester in an HNE buffer for 45 min at room temperature. The unincorporated dye was separated from fluorescence-labeled ZIKV particles by a Sephadex G-25 column (GE Healthcare). The fractions containing Atto647N-labeling ZIKV were detected by a multimode microplate reader (TECAN 200/200Pro) and stored at −80°C.
10
Production and Purification of Influenza Virus-Like Particles
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