Cells in a 10 cm plate were lysed in 2 mL of cell lysis buffer (10 mM Tris-HCl, pH7.5, 1 mM EDTA, 0.5% NP40) at room temperature for 10 min. The cell debris was cleared by centrifugation at 12,000× g for 10 min at 4 °C. The supernatant was loaded onto a 30% sucrose cushion and centrifuged at 46, 000 rpm for 3.5 h (Beckman, Indianapolis, IN, USA; SW55 rotor). The pellet was dissolved in 200 µL TNE buffer containing protease inhibitor (5892953001, Roche, Basel, Switzerland) overnight, then loaded onto a 15–50% linear sucrose gradient in TNE buffer and centrifuged at 27,000 rpm for 8 h (Beckman, Indianapolis, IN, USA; SW55 rotor). Fractions (250 µL/each) were collected from the top of the centrifugation tube. A total of 25 µL of each fraction was applied to agarose gel electrophoresis or SDS-PAGE to detect viral capsids or HBc.
Sw55 rotor
Manufactured by Beckman Coulter
Sourced in United States
The SW55 rotor is a high-speed centrifuge rotor designed for use with Beckman Coulter ultracentrifuges. The rotor has a maximum speed of 55,000 rpm and can achieve a maximum RCF (Relative Centrifugal Force) of 330,000 x g. The SW55 rotor is compatible with a variety of sample tubes and is commonly used for applications such as gradient separation and subcellular fractionation.
Automatically generated - may contain errors
Lab products found in correlation
Ty 50.2 rotor, by Beckman Coulter (4 mentions)
Gradient master, by BioComp Instruments (3 mentions)
Sw28 rotor, by Beckman Coulter (3 mentions)
Sw32ti rotor, by Beckman Coulter (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Gradient fractionator system, by Brandel Inc (2 mentions)
Ua 6 absorbance detector, by Teledyne (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Proteinase inhibitor cocktail, by Merck Group (2 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
0.45 μm pore size filter, by Merck Group (1 mentions)
Hiv 1 p24 elisa kit, by Sino Biological (1 mentions)
Uv crosslinker, by Spectroline (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Pka enzyme, by New England Biolabs (1 mentions)
Amicon centrifugal filter unit, by Merck Group (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Protease inhibitors cocktail, by Merck Group (1 mentions)
Nupage 4 12 bis tris sds page, by Thermo Fisher Scientific (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
80 protocols using sw55 rotor
1
Purification of Viral Capsids
2
Generation of Filovirus-Pseudotyped HIV Particles
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Pseudo-filoviruses were produced as previously described11 , 13 (link). Briefly, the plasmids encoding filovirus-GP and the HIV vector (pNL4.3.Luc-R−E−) were co-transfected at a 1:1 ratio into 293T producer cells in a 100-mm culture dish using jetPRIME transfection reagent according to the manufacturer's protocol (Polyplus-transfection). Forty-eight hours post-transfection, the HIV/filovirus-GP pseudovirions containing cell culture supernatants were collected and filtered through a 0.45 μm pore size filter (Millipore). Then, for the in vivo study, the pseudoviruses were layered onto a cushion of 20% (w/v) sucrose and centrifuged at 50,000 rpm for 2 h in a Beckman SW55 rotor at 4 °C. The pelleted pseudoviruses were re-suspended in ice-cold NTE buffer (10 mmol/L Tris, 100 mmol/L NaCl, 1 mmol/L EDTA, pH 7.5) and stored at –80 °C until use. HIV-1 p24 protein in the viral particles was detected by an HIV-1 p24 ELISA kit (Sino Biological Inc.) according to the manufacturer's protocol.
3
Probing mRNA Pathway in Initiation Complexes
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To examine the effect of DHX29 on the pathway taken by mRNA in the initiation complexes, we used a UV-cross-linking assay. The 48S ICs were reconstituted with/without DHX29 in an 80 µL reaction mixture with scaled amounts of the initiation components on co-transcriptionally 32P-labeled and 4-thioU-incorporated “+5,” “+7,” “+9,” “+11,” “+13,” and “+15” mRNAs. After assembly, the initiation complexes were purified from unbound components by centrifugation through 10%–30% SDG prepared in buffer A in a Beckman SW55 rotor at 53,000 rpm for 75 min, irradiated at 360 nm for 30 min on ice using a UV Crosslinker (Spectroline), and digested with 5 units RNase A for 10 min at 37°C. The samples were assayed by electrophoresis in NuPAGE 4%–12% Bis-Tris gel (Thermo Fisher Scientific) and autoradiography. After the RNase treatment of the samples, the cross-linked proteins acquired additional weight in the form of nucleotides, which resulted in a shifted band on SDS-PAGE. For the UV-cross-linking experiment with the eIF3/mRNA binary complex, we repeated this protocol but omitted the SDG purification step.
4
Ribosomal Association of DHX29 Isoforms
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PKA-DHX29 (FL), PKA-DHX29 (983–1369), and PKA-DHX29 (1–1227) in Ni-NTA eluates were [32P]-labeled with PKA enzyme (NEB) and [γ-32P]ATP. To study the ribosomal association of different forms of DHX29, the 43S PICs assembled in a 400-µL reaction mixture with scaled amounts of initiation components were supplemented with 50 pmol DXH29 (1–594) or 100 µL Ni-NTA eluate containing [32P]-labeled DHX29, purified from unbound components by centrifugation through 10%–30% SDG prepared in buffer A in a Beckman SW55 rotor at 53,000 rpm for 75 min, resolved in SDS-PAGE, and analyzed by immunoblotting (DHX29 [1–594]) or autoradiography ([32P]-labeled forms of DHX29).
5
Purification of Human Metapneumovirus for m6A Sequencing
6
Liposome Co-flotation Assay for Protein Binding
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Liposome co-flotation assay was carried out using a previously established procedure (Shen et al., 2007 (link); Yu et al., 2018 (link)). Soluble proteins were incubated with protein-free or v-SNARE liposomes at 4°C with gentle agitation. After one hour, an equal volume of 80% (w/v) Nycodenz was added and the samples were transferred to 5 × 41 mm centrifuge tubes. The samples were overlaid with 200 μL each of 35% (w/v) and 30% (w/v) Nycodenz, and then with 20 μL reconstitution buffer on the top. All Nycodenz solutions were prepared in the reconstitution buffer. After centrifugation at 52,000 rpm for four hours in a Beckman SW55 rotor, samples were collected from the 0/30% Nycodenz interface and analyzed by SDS-PAGE.
7
Velocity Gradient Centrifugation of Protein Samples
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For velocity gradient centrifugation, samples (∼0.2 ml, ∼1 mg) were loaded onto a 4.6-ml linear 10–30% (wt/vol) sucrose gradient in 20 mM HEPES with 1 mM EDTA and the protease and phosphatase inhibitor cocktail, pH 7.4, and centrifuged for 55 min in a SW55 rotor (Beckman Coulter, Fullerton, CA) at 48,000 rpm. Each gradient was separated into 22–26 fractions starting from the bottom of the tube. Fractions were further analyzed by SDS–PAGE and Western blotting.
8
Purification of Human Metapneumovirus for m6A Sequencing
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Parental hMPV strain NL/1/00 was propagated and titrated in Vero E6 cells. To prepare highly purified hMPV for m6A sequencing, twenty T150 flasks of A549 cells were infected by hMPV at an MOI of 0.5. Cell culture supernatants harvested at 72 h post-infection were clarified by centrifugation at 5,000 ×g for 30 min. Virus was concentrated by centrifugation at 30,000×g for 2 h at 4°C in a Ty 50.2 rotor (Beckman). The pellet was resuspended in NTE buffer (0.05 M Tris-HCl, 0.15 M NaCl, 15 mM CaCl2 [pH 6.5]) supplemented with 10% trehalose and further purified through a sucrose gradient by centrifugation at 35,000×g for 18 h at 4°C in an SW55 rotor (Beckman). Solution layer containing virus was extracted with syringe, diluted with NTE buffer and centrifuged at 30,000×g for 2 h at 4°C in SW55 rotor. The final pellet was resuspended in 0.5 ml of NTE buffer.
9
Phosphorylation of Eukaryotic 60S Ribosomal Subunit
10
Sucrose Gradient Centrifugation of Proteins
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The sucrose gradient centrifugation experiments were performed as described (Martinelli et al., 2012 (link)). Briefly the indicated proteins were separated on sucrose gradients in HBS buffer (0.01 M Hepes, pH 7.4, 0.15 M NaCl), by overlaying sucrose solutions of 60% (65 μl), 40%, 30%, 20% and 5% (85 μl each). Centrifugation was performed in a Beckman SW55 rotor at 40,000 rpm for 6h at 4°C. Fractions from the gradients were analysed on a 15% SDS-PAGE and bands were detected with Coomassie Blue staining.
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