Ribosome–polysome profile analysis was performed as described previously (16 (link)) with modifications. Namely, twenty A260 units of extract were layered onto 7–47% sucrose (w/w) gradient and centrifuged at 4°C in SW28 rotor (Beckman Coulter, Brea, CA, USA) at ω2t = 1.8 × 1011. For 60S and 40S ratio quantification ribosome-polysome profile analysis was carried out with following modifications. The addition of cycloheximide to the cell cultures was omitted and Mg2+ was excluded from all buffers and sucrose gradients. Twenty A260 units of extract were layered onto 10–25% sucrose (w/w) gradient and centrifuged at 4°C in SW28 rotor (Beckman Coulter) at ω2t = 3.4 × 1011. Areas under monosome (80S), polysome, 60S and 40S peaks were quantified by ImageJ and corresponding ratios were calculated. The average and standard deviations for at least three biological replicates were calculated. Statistical significance was determined by the unpaired two sample Student's t test.
Sw28 rotor
Manufactured by Beckman Coulter
Sourced in United States, Australia
The SW28 rotor is a high-speed ultracentrifuge rotor designed for the separation and purification of macromolecules, organelles, and particles. It is capable of achieving centrifugal forces up to 150,000 x g, making it suitable for a wide range of applications in the fields of molecular biology, biochemistry, and cell biology.
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Lab products found in correlation
Sw41 rotor, by Beckman Coulter (8 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Sw60ti rotor, by Beckman Coulter (7 mentions)
Econo uv monitor, by Bio-Rad (7 mentions)
Optima l 100 xp ultracentrifuge, by Beckman Coulter (6 mentions)
Pspax2, by Addgene (6 mentions)
Fc203b, by Gilson (6 mentions)
Optiprep, by Merck Group (5 mentions)
Ultra clear tube, by Beckman Coulter (5 mentions)
Elisa for p24, by PerkinElmer (4 mentions)
Sw40 rotor, by Beckman Coulter (4 mentions)
Sw55ti rotor, by Beckman Coulter (4 mentions)
Padvantage, by Promega (4 mentions)
Tla 55 rotor, by Beckman Coulter (4 mentions)
Pcmv vsv g, by Addgene (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Exosome depleted fbs, by System Biosciences (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Total exosome rna protein isolation kit, by Thermo Fisher Scientific (3 mentions)
260 protocols using sw28 rotor
1
Ribosome-Polysome Profile Analysis
2
Purification of Baculovirus-Derived VLPs
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Baculovirus infected H5 cell monolayers were harvested after incubation for 4 days at 28 °C, washed three times with 0.2 M phosphate-buffered saline for VLPs (PBS-V: 0.2 M sodium phosphate, 0.1 M NaCl, pH 6.0) and pellets were resuspended in distilled water. After mild sonication and treatment with DNAse I (Roche, Basel, Switzerland), samples were adjusted to 2% Sarkosyl (sodium N-lauroylsarcosine, Sigma), 5 mM EDTA in PBS-V, and incubated overnight at 4 °C. Next, cell lysates were clarified by low-speed centrifugation and the supernatants were centrifuged using a Beckman SW28 rotor at 27,000 rpm for 2 h. Pellets were resuspended in PBS-V, extracted with Vertrel XF (Fluka, Sigma-Aldrich, St. Louis, MO, USA), and centrifuged using a Beckman SW28 rotor at 27,000 rpm for 2 h. The pelleted material was subjected to centrifugation through a 20% (wt/vol) sucrose cushion in PBS-V at 35,000 rpm for 2.5 h using a Beckman SW55 rotor. Finally, the pellets were resuspended in PBS-V containing protease inhibitors (Complete, Roche, Penzberg, Germany) and stored at 4 °C. Protein concentrations of the VLP samples were determined using BCA protein assay kit, Pierce, Thermo Scientific (Waltham, MA, USA).
Recombinant protein expression was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE).
Recombinant protein expression was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE).
3
Production and Purification of Virus-Like Particles
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H5 cell monolayers were infected with recombinant baculoviruses at a multiplicity of infection (MOI) of 10. After incubation (4 days, 28 °C), infected cells were dislodged into the growth medium and collected. Suspensions were then washed three times with 0.2 M phosphate-buffered saline for VLPs (PBS-V, consisting of 0.2 M sodium phosphate, 0.1 M NaCl, pH 6.0). Next, pellets were resuspended in distilled water, subjected to mild sonication and treated with DNAse I (Roche Applied Science) for 1 h at room temperature. Subsequently samples were adjusted to 2% Sarkosyl (sodium N-lauroylsarcosine, Sigma), 5 mM EDTA in PBS-V, and incubated overnight at 4 °C. Cell lysates were then clarified by low-speed centrifugation and the supernatant was centrifuged at 27,000 rpm for 2 h using a Beckman SW28 rotor. The pelleted material was resuspended in PBS-V, extracted 2–3 times with Vertrel XF (Fluka, Sigma-Aldrich), and centrifuged at 27,000 rpm for 2 h using a Beckman SW28 rotor. The pellets were finally resuspended in PBS-V containing protease inhibitors (Complete, Roche) and stored at 4 °C. Protein concentrations of the VLP preparations were determined with BCA protein assay kit, Pierce, Thermo Scientific.
4
Extracellular Vesicle Isolation Protocol
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Like for the filter fractionation, conditioned media was cleared of cells as above and EVs from the cleared media were prepared by ultracentrifugation, first at 10,000 ×g (8,700 rpm in SW28 rotor, Beckman Coulter), then the supernatant of that spin at 100,000 ×g (27,000 rpm in SW28 rotor, Beckman Coulter). The pellet for the 10,000 ×g spin was combined by spinning at 13,000 rpm in a bench-top micro-centrifuge and the pellet for the 100,000 ×g spin was washed once with PBS and re-pelleted at 100,000 ×g.
5
Extracellular Vesicle Isolation Protocol
6
Generating Herpes Virus Mutant Stocks
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Stocks of the UL25 deletion mutant (UL25-null) (36 (link)) were generated by using a complementary African green monkey cell line (both gifts from Fred L. Homa, University of Pittsburgh, Pittsburgh, PA). At roughly 75% confluence, monolayers in 175-cm2 flasks were infected with virus stock at a multiplicity of infection (MOI) of 0.1 in 1 ml of phosphate-buffered saline (PBS) without calcium or magnesium per flask for 40 min at room temperature, followed by 5 min at 37°C. The cells were then overlaid with 15 ml virus growth medium (1× minimal essential medium [MEM] Alpha, 1.5% [vol/vol] penicillin-streptomycin; Corning) and incubated at 37°C. At 48 h postinfection, virus-containing medium was clarified by centrifugation at 1,500 rpm for 5 min, and crude cell debris were removed from the supernatant by pelleting at 6,000 rpm for 15 min in a Beckman SW28 rotor. Virus was pelleted for 45 min at 19,000 rpm in a Beckman SW28 rotor. The virus pellets were resuspended in small volumes of PBS without calcium or magnesium, and aliquots of 500 μl were frozen at −80°C. The virus titer was assessed in complementary cells 48 h after infection.
Stocks of the procapsid-producing M100 line (35 (link)) were generated similarly using the complementary F-3 cell line. Stock titers were assessed as described earlier (5 (link)).
Stocks of the procapsid-producing M100 line (35 (link)) were generated similarly using the complementary F-3 cell line. Stock titers were assessed as described earlier (5 (link)).
7
Milk Extracellular Vesicle Isolation by Differential Centrifugation
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Differential centrifugation and ultracentrifugation were used for the isolation of EVs from various raw milk samples, as previously described [1 (link)]. Briefly, the defatted milk samples were subjected to differential centrifugation (210 mL starting milk volume in SW-28 rotor, Beckman Coulter, 35 mL open top tubes, Life Sciences, Boston, MA, USA) starting at 5000× g for 30 min at 4 °C. The fat layer floating on top was removed carefully. After the removal of fat globules and milk-abundant proteins such as casein and cell debris, the skimmed milk samples were subjected to successive centrifugations at 4 °C for 1 h each at 12,000× g, 35,000× g and 70,000× g. Lastly, the supernatant obtained following these spins was centrifuged at 100,000× g using a SW-28 rotor (Beckman Coulter instruments, Waverley, VIC, Australia). The pellet obtained, containing enriched milk EVs, was resuspended in sterile, filtered PBS. A final washing step was performed using ultracentrifugation at 100,000× g for 1 h at 4 °C (Beckman Coulter: TLA-55 rotor). The resulting EV pellet obtained with this method was resuspended in the appropriate volume of PBS and stored at −80°C until further analysis.
8
Isolation and Characterization of Small Extracellular Vesicles from PBDE-Treated THP-1 Macrophages
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The THP-1 monocytes were seeded at a concentration of 6x105 cells/mL in 75 cm2 flasks and differentiated in M(0) THP-1. Then, cells were washed with micro-filtered (0.2 μm pore size) 1X PBS w/o Ca2+ and Mg2+ and incubated with 0.0125% DMSO (Control) or 3 μM PBDE-47 in RPMI medium with 10% FBS previously depleted of microvesicles by centrifugation at 118,000 g at 4°C overnight using a Beckman SW28 rotor. Thereafter, the M(0) THP-1 macrophages were differentiated in M(LPS) THP-1 for 24 hours at 37°C in 5% CO2. The supernatants containing DMSO derived (sEVsDMSO) and PBDE derived (sEVsPBDE) small extracellular vesicles were first centrifuged at low speed to remove cells and debris. The large EVs were then isolated by centrifugation at 10,000 x g for 30 minutes at 4°C using an Eppendorf rotor F34-6-38 and resuspended in a proper volume of 1X PBS w/o Ca2+ and Mg2+. Afterwards, sEVs were collected from the supernatants into Beckman Coulter polypropylene open top tubes via centrifugation at 118,000 g for 70 minutes at 4°C using a Beckman SW28 rotor. Finally, the sEVs were washed in 1X PBS w/o Ca2+ and Mg2+ and resuspended in the same buffer.
9
Exosome Purification from Cell Lines
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For the purification of exosomes, Akata−, Akata+, Akata-LCLs (1 × 107 cells, each) were grown in RPMI 1640 medium containing 10% exosome-depleted FBS, which was prepared by centrifugation at 112,400 × g for 4 h at 4°C with an SW28 rotor (Beckman Fullerton, USA). Culture medium containing exosomes was harvested and centrifuged at 1,500 × g for 10 min and at 6,000 × g for 20 min to remove cells and cell debris, respectively. The exosomes were pelleted by centrifugation at 112,400 × g for 1 h at 4°C with an SW28 rotor (Beckman). The pelleted exosomes were resuspended in TNE buffer [10 mM Tris-HCl (pH 7.6), 100 mM NaCl, 1 mM EDTA] overnight. The fractions containing exosomes were confirmed by western blot analysis with anti-CD63 monoclonal antibody (clone MEM-250, Abnova, Taipei, Taiwan).
10
Isolation and Purification of E. coli Ribosomes
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E. coli BL21 cells were harvested from 1L culture in LB media (A600 ~1.5), and the cells were lysed rotor. Pellets were gently dissolved in a small volume of monosome gradient buffer and the concentration was approximated using 1 A260 unit = 24 pmol of ribosomes per mL. Approximately 200 A260 units of crude E.coli BL21 ribosome isolate (~0.5 mL) was adjusted to 1 mL with monosome gradient buffer and carefully loaded onto a 10-50% gradient of sucrose in monosome buffer in SW-28 tubes (for Beckman SW-28 rotor). The samples were centrifuged for 20 hours in a Beckman SW-28 rotor at 18000 rpm, 4°C. The resulting gradients were harvested at 4°C using a peristaltic pump and fractions of ~1 mL were collected. Fractions containing the 70S ribosomes were pooled and pelleted by ultracentrifugation in the Ti70.1 rotor (Beckman) for 3 h at 48000 rpm, 4°C. The pellet was gently dissolved in 500 μL of monosome buffer, aliquoted in portions of 50 μL, flash frozen in liquid nitrogen and stored at -80°C.
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