Insect cells expressing full length Atg9 were lysed by passing the cell suspension through an EmulsiFlex. The lysate was cleared using a low speed spin (9000 g for 30 minutes), followed by centrifugation of the resulting supernatant at 40,000 rpm for 1 hour (Ti-45 rotor; Beckman). Pelleted membranes were resuspended in solubilization buffer (50 mM Tris HCl pH 8.0, 300 mM NaCl, 5% glycerol, 2 mM DTT) containing 1% n-Dodecyl-β-D-Maltopyranoside (DDM). The sample was incubated for 4 hours at 4°C before centrifuging at 40,000 rpm for 1 hour (Ti-45 rotor; Beckman). The supernatant was loaded onto a StrepTactin column pre-equilibrated in wash buffer (50 mM Tris HCl pH 8.0, 300 mM NaCl, 2 mM DTT containing either 0.6 mM DDM or 0.6 mM Lauryldimethylamine-N-Oxide (LDAO)). The column was washed with 10 CV of wash buffer and the protein was eluted using wash buffer containing 2.5 mM desthiobiotin. StrepTactin elutions were concentrated and used directly.
Ti45 rotor
Manufactured by Beckman Coulter
Sourced in United States
The Ti45 rotor is a high-speed centrifuge rotor designed for Beckman Coulter ultracentrifuges. It is capable of reaching speeds up to 45,000 rpm and can generate relative centrifugal forces up to 286,000 x g. The Ti45 rotor is commonly used for a variety of applications, such as the separation and purification of macromolecules, organelles, and other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor, by Roche (3 mentions)
Hiprep, by Cytiva (3 mentions)
Sw32 rotor, by Beckman Coulter (2 mentions)
Sw56 rotor, by Beckman Coulter (2 mentions)
Tla 55 rotor, by Beckman Coulter (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Cell disruptor, by Constant Systems (2 mentions)
Dnase, by Merck Group (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Histrap 5 ml, by GE Healthcare (2 mentions)
High five insect cells, by Thermo Fisher Scientific (1 mentions)
Ni nta resin, by Qiagen (1 mentions)
Native page 4 16 bis tris protein gels, by Thermo Fisher Scientific (1 mentions)
Total exosome isolation kit, by Thermo Fisher Scientific (1 mentions)
Optima xpn 100, by Beckman Coulter (1 mentions)
Superose 6 increase 3.2 300 column, by GE Healthcare (1 mentions)
Glutathione sepharose, by GE Healthcare (1 mentions)
Mf millipore membrane filter, by Merck Group (1 mentions)
Dls system, by Wyatt Technology (1 mentions)
α thioglycerol, by Merck Group (1 mentions)
54 protocols using ti45 rotor
1
Purification of Full-Length Atg9 Protein
2
Ribosome Isolation from Thermoacidophilic Archaeon
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Ribosomes were prepared from the archaeon S. acidocaldarius (Sac) DSM639, as follows. Cells were initially grown at 75°C and pH 3.5. To obtain Sac ribosomes, 2g samples of cell pellets were harvested and resuspended in 30 ml of buffer A (25 mM Tris–HCl pH 7.5, 100 mM NH4Cl, and 10.5 mM MgCl2) at 4°C, lysed twice (18 kPa; ConstantSystem), and centrifuged at 16,300g (HITACHI R20A2) for 40 min. The supernatants thus obtained were loaded onto a 37.7% sucrose cushion (20 mM Tris–HCl pH 7.5, 37.7% sucrose, 100 mM NH4Cl, and 10.5 mM MgCl2) and centrifuged in a Ti45 rotor (Beckman) at 167,900g for 21 h at 4°C. Subsequently, the pellets were suspended in buffer A, loaded onto a 10–35% sucrose gradient, and spun in an SW32 rotor (Beckman) at 68,300g for 13 h at 4°C. Fractions containing the 30S subunits were collected using a Biocomp Piston Gradient Fractionator and pelleted separately after centrifugation in a Ti45 rotor (Beckman) at 167,900g for 17 h at 4°C. Finally, the pellets were suspended in buffer B (25 mM HEPES KOH pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, and 1 mM DTT), flash-frozen in liquid nitrogen, and stored at −80°C until further use.
3
Purification of recombinant P-glycoprotein
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Crude membranes from High Five insect cells (Thermo Fisher Scientific, Waltham, MA) expressing wild-type (WT)–hP-gp, or its double mutant EQ–hP-gp (E556Q/E1201Q), with a 6-histidine tag at the C-terminal end were isolated as described previously (Kerr et al., 2001 (link)). The membranes (150–250 mg of protein) were solubilized by using 2% n-dodecyl β-d -maltoside in a buffer containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 15% glycerol, 5 mM β-mercaptoethanol, 20 mM imidazole, and UltraCruz EDTA-free protease inhibitor cocktail tablets (Santa Cruz Biotechnology, Dallas, TX). The solubilized extract was centrifuged at 38,000 rpm (Ti-45 rotor; Beckman Coulter, Brea, CA) for 45 minutes at 4° C. The supernatant was incubated with Ni-NTA resin (Qiagen Inc., Valencia, CA) pre-equilibrated in solubilization buffer with 0.09% n-dodecyl β-d -maltoside for 14–16 hours at 4° C. The beads were washed and P-gp was eluted with the same buffer containing 300 mM imidazole. Fab isolated from UIC2 monoclonal antibody was then added at a molar ratio of P-gp:Fab (1:3) and incubated at 4° C for 15 minutes. The hP-gp–Fab complex was centrifuged at 300,000 × g for 45 minutes. The complex formation was evaluated on native PAGE 4–16% Bis-Tris protein gels (Thermo Fisher Scientific, Waltham, MA).
4
Exosome Isolation from Conditioned Media
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Cell culture media was harvested from adMSCs, grown initially in the presence of 10% foetal bovine serum (FBS) and without FBS for the last 12 h. After 12 h, cells and debris were removed by centrifugation at 350×g for 10 min and 2000×g for 30 min. For ultracentrifugation, the samples were pre-enriched at 100,000×g for 70 min at 4 °C using a Ti45 rotor (Beckman) and Beckman ultracentrifugation tubes (355622) on a Beckman Optima XPN-100. For precipitation, Total Exosome Isolation kit (Thermo Fisher Scientific, USA) was added to the cell and debris-free cell medium (1:2 with exosome isolation reagent and cell medium, respectively). Cell medium and the exosome isolation reagent were mixed by brief vortexing and incubated at 4 °C overnight before centrifugated at 4 °C at 10,000×g for 1 h. The pellet containing pre-enriched exosomes was resuspended in PBS [39 ].
5
Norovirus Virus-Like Particle Production
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Full-length VP1 genes for GI.1 West Chester, GII.4 Saga 2006, GII.10 Vietnam, GII.17 Kawasaki308, and GII.17 Saitama T87 (GenBank accession numbers: AY502016.1, AB447457.1, AF504671.2, LC037415.1, AII73747.1) were cloned and expressed in a baculovirus system [29 (link),30 (link)]. After transfection of a bacmid containing the recombinant VP1 gene in Sf9 insect cells and incubation for 5–7 days, the culture medium was collected and centrifuged for 10 min at 3000 rpm at 4 °C. Subsequently, Hi5 insect cells were infected with recovered baculovirus and incubated for 5 days. After centrifuging the culture medium for 10 min at 3000 rpm at 4 °C and then 1 h at 6500 rpm at 4 °C, VLPs in the supernatant were concentrated by ultracentrifugation at 35,000 rpm (Beckman Ti45 rotor, Krefeld, Germany) for 2 h at 4 °C. Furthermore, VLPs were further purified using CsCl equilibrium gradient ultracentrifugation at 35,000 rpm (Beckman SW56 rotor, Krefeld, Germany) for 18 h at 4 °C. VLPs were pelleted for 2 h at 40,000 rpm (Beckman TLA55 rotor, Krefeld, Germany) at 4 °C and solved in PBS (pH 7.4).
6
Purification of ATP Synthase Dimers
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Approximately 10 mg mitochondria were lysed in a total volume of 90 ml buffer C (25 mM HEPES/KOH pH 7.5, 25 mM KCl, 15 mM MgOAc2, 1.7% Triton-X100, 2 mM DTT, one tablet EDTA-free Protease Inhibitor Cocktail) for 2 hr at 4°C and the lysate was cleared by centrifugation at 30,000 xg, 20 min, 4°C. The supernatant was layered on a sucrose cushion in buffer D (1 M sucrose, 25 mM HEPES/KOH pH 7.5, 25 mM KCl, 15 mM MgOAc2, 1% Triton-X100, 2 mM DTT) and centrifuged 158,420 xg, 3 hr, 4°C in a Ti45 rotor (Beckman Coulter). The resulting pellet was resuspended in 200 µl buffer E (25 mM HEPES/KOH pH 7.5, 25 mM KCl, 15 mM MgOAc2, 2 mM DTT, 0.05% β-DDM) and gel filtrated over a Superose 6 Increase 3.2/300 column (GE Healthcare) in buffer E. Fractions corresponding to ATP synthase dimers were pooled and concentrated to 25 µl in a vivaspin500 filter (100 kDa MWCO).
7
Purification of GST-tagged Proteins
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GST‐tagged proteins were expressed in E. coli BL21 cultured in terrific broth media at 37°C for 2 h following induction with 0.15 mM IPTG at OD600 0.5‐0.7. The bacteria were harvested and lysed with a high‐pressure homogeniser (Constant Systems) in 50 mM HEPES pH 7.4, 500 mM NaCl, 2 mM DTT. The lysate was cleared by centrifugation in a Beckman Ti45 rotor at 30000 rpm for 25 min at 4°C. The supernatant was incubated with glutathione sepharose (GE Healthcare) for 10 min, spun down, washed extensively, and the beads were finally resuspended in 20 mM HEPES pH 7.4, 150 mM NaCl and 0.5 mM TCEP and used in pull‐down experiments.
8
Saliva-Derived Extracellular Vesicle Isolation and Pooling
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Each saliva sample was diluted 1:1 with phosphate-buffered saline (PBS) prior to proceed to SEV isolation following the experimental workflow shown in Figure 1 B. As reported, the saliva samples were centrifuged at 300× g for 5 min at 30 °C to eliminate the cells. Then, the supernatant was centrifugated at 3000× g for 15 min at 4 °C, and further at 10,000× g for 30 min at 4 °C to eliminate cell debris, other contaminants, and M/LEVs as well. Finally, the supernatant was filtered through a 0.45 μm VWR® Vacuum Filtration System (VWR International, West Chester, PA, USA), before ultracentrifugation (Ti70 or Ti45 rotor, Beckman Coulter, Brea, CA, USA) at 100,000× g for 70 min at 4 °C to pellet the SVEs, which were finally resuspended in 100 µL PBS.
In order to improve the protein amount and also to minimize the individual-to-individual differences, the SEV pellets isolated from saliva samples (S/SEVs) of the same group were pooled. Thus, subsequent analyzes were carried out on three types of pooled samples: (a) S/SEV OSCC_FREE; (b) S/SEV OSCC_NLNM; and (c) S/SEV OSCC_LNM.
In order to improve the protein amount and also to minimize the individual-to-individual differences, the SEV pellets isolated from saliva samples (S/SEVs) of the same group were pooled. Thus, subsequent analyzes were carried out on three types of pooled samples: (a) S/SEV OSCC_FREE; (b) S/SEV OSCC_NLNM; and (c) S/SEV OSCC_LNM.
9
Exosome Isolation from Cultured Stem Cells
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hESCs and hiPSCs were cultured in ncTarget medium (cat. no. RP01020; Nuwacell. Ltd, China), while the 3rd generation of hUC-MSCs were cultured in serum-free ncMission hMSC medium (cat. no. RP02010; Nuwacell. Ltd, China). Next, 350 mL of the supernatant of cells in the logarithmic growth phase (~ 80% confluency) was collected for exosome purification. The exosomes were extracted in accordance with a series of recognized centrifugation and ultracentrifugation, as previously described [34 (link), 52 (link)]. Briefly, the conditioned media (CM) were collected and subjected to gradient centrifugation (300 × g for 10 min, 2000 × g for 15 min, 10, 000 × g for 30 min) to separate cell debris. Exosomes were pelleted from the collected supernatants at 100, 000 × g for 70 min using a Ti45 rotor (Beckman Coulter, USA). They were then resuspended in PBS for the next filtration using a 0.22-μm MF-Millipore™ Membrane filter (Sigma, USA). The filtrate was subjected to ultracentrifugation at 100, 000 × g for 70 min. The final exosome pellet was resuspended in PBS for the next analysis. Dynamic light scattering (DLS) system (Wyatt Technology, USA) was used to measure the concentration and size of the isolated exosomes.
10
Culturing Human Mast and Erythroleukemic Cells
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The human mast cell line HMC-1 (Dr. Joseph Butterfield, Mayo Clinic, Rochester, MN, USA) and the erythroleukemic cell line TF-1 (ATCC: CRL-2003) were cultured in a 37°C humidified incubator with 5% CO2. HMC-1 was cultured in Iscove's Modified Dulbecco's Medium (IMDM), while TF-1 was cultured in RPMI-1640. Both media were supplemented with 10% foetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L -glutamine. In addition, the medium for HMC-1 also contained 1.2 mM α-thioglycerol and the medium for TF-1 was supplemented with 5 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF) (all reagents were from Sigma-Aldrich, St Louis, MO, USA).
To eliminate serum exosomes, the FBS was ultracentrifuged (Ti45 rotor, Beckman Coulter, Brea, CA, USA) for 18 hours at 120,000 × g (average) prior to use in cultures.
The cell viability was calculated using the trypan blue dye method.
To eliminate serum exosomes, the FBS was ultracentrifuged (Ti45 rotor, Beckman Coulter, Brea, CA, USA) for 18 hours at 120,000 × g (average) prior to use in cultures.
The cell viability was calculated using the trypan blue dye method.
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