Wild-type and recombinant novirhabdoviruses were mass produced in EPC cells, clarified by low-speed centrifugation (4,000 rpm for 15 min), concentrated 10 fold by ultracentrifugation at 24,000 rpm in a SW28 Beckman rotor for 90 minutes and finally purified by ultracentrifugation at 34,000 rpm in a SW41 Beckman rotor for 4 hours through a 25% (w/v) sucrose cushion in TEN buffer (10 mM Tris-HCl [pH = 7.5], 150 mM NaCl, 1 mM EDTA [pH = 8]). Influenza virus were mass produced in MDCK cells, inactivated by UV-illumination at 254 nm for 5 minutes (UVitec) and then purified by ultracentrifugation at 34,000 rpm in a SW41 Beckman rotor for 4 hours through a 25% (w/v) sucrose cushion. Virus pellets were resuspended in TEN buffer and the viral protein yield of each preparation was quantified by using the Micro BCA assay protein quantification Kit (Pierce) in accordance with the manufacturer’s instructions.
Beckman sw41 rotor
Manufactured by Beckman Coulter
Sourced in United States
The Beckman SW41 Rotor is a high-performance, swinging-bucket centrifuge rotor designed for a variety of laboratory applications. It can achieve maximum speeds of up to 275,000 x g, enabling efficient separation and isolation of particles, cells, and macromolecules. The rotor features a durable and corrosion-resistant construction, making it a reliable choice for routine laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Beckman sw28 rotor, by Beckman Coulter (2 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Isogen ls kit, by Nippon Gene (1 mentions)
Optima le 80k, by Beckman Coulter (1 mentions)
Prism 7, by GraphPad (1 mentions)
Rnasin plus rnase inhibitor, by Promega (1 mentions)
Polycarbonate centrifuge tube, by Beckman Coulter (1 mentions)
Brij o10, by Merck Group (1 mentions)
Gradient master, by BioComp Instruments (1 mentions)
Ua 6 uv vis detector, by Teledyne (1 mentions)
Beckman sw 55 rotor, by Beckman Coulter (1 mentions)
18 protocols using beckman sw41 rotor
1
Purification and Quantification of Viruses
2
Isolation of Lipid Rafts from Cells
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C ells were washed twice with ice-cold PBS, scraped into 2 mL of 500 mM sodium carbonate (pH 11.0), transferred to a plastic tube, and homogenized with a Sonicator 250 apparatus (Branson Ultrasonic, Danbury, CT) using three 20-sec bursts. The homogenate was adjusted to 45% sucrose by the addition of 2 mL 90% sucrose prepared in 2-(N-morpholino) ethanesulfonic acid (MES)-buffered solution consisting of 25 mM MES-buffer solution (pH 6.5) and 0.15 M NaCl and placed at the bottom of an ultracentrifuge tube. A 5–35% discontinuous sucrose gradient was formed (4 mL each of 5% and 35% sucrose, both in MES-buffer solution containing 250 mM sodium carbonate) and centrifuged at 40,000 × g for 20 h in a Beckman SW41 Rotor (Beckman Coulter, Fullerton, CA). Eight fractions were collected and analyzed by 12% SDS-PAGE.
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3
Sucrose Density Gradient Centrifugation
4
Sucrose Gradient Analysis of Testicular Ribosomes
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Sucrose gradient analysis was carried out as described previously with minor modifications [34 (link), 36 (link)]. Briefly, testicular tissues (~0.1 g)
were homogenized at 4°C in 1 ml of HK buffer (20 mM HEPES/KOH, pH 7.4, 0.1 M KCl) containing 5 mM MgCl2, 0.5% Triton X-100, 0.2 mg/ml cycloheximide, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 0.5 mM PMSF, and 40
U/ml RNaseOUT (Thermo Fischer Scientific) and centrifuged at 13,400 × g for 10 min at 4°C. The supernatant solution (0.9 ml) was layered onto a 10–45% sucrose gradient (9.5 ml) prepared in HK buffer
containing 5 mM MgCl2 and centrifuged in a Beckman Optima LE-80K ultracentrifuge using a Beckman SW-41 rotor (Beckman Coulter, Fullerton, CA, USA) at 281,000 × g at 4°C for 2 h. Fractions
(approximately 1 ml each) were manually collected from the top of the gradient and RNA levels in the fractions were measured by a UV spectrophotometer at 260 nm (Nanodrop, Thermo Fischer Scientific). Aliquots of each
fraction were analyzed by immunoblotting or by ethidium bromide staining of ribosomal RNAs (rRNAs) purified using an ISOGEN LS kit (Nippon Gene).
were homogenized at 4°C in 1 ml of HK buffer (20 mM HEPES/KOH, pH 7.4, 0.1 M KCl) containing 5 mM MgCl2, 0.5% Triton X-100, 0.2 mg/ml cycloheximide, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 0.5 mM PMSF, and 40
U/ml RNaseOUT (Thermo Fischer Scientific) and centrifuged at 13,400 × g for 10 min at 4°C. The supernatant solution (0.9 ml) was layered onto a 10–45% sucrose gradient (9.5 ml) prepared in HK buffer
containing 5 mM MgCl2 and centrifuged in a Beckman Optima LE-80K ultracentrifuge using a Beckman SW-41 rotor (Beckman Coulter, Fullerton, CA, USA) at 281,000 × g at 4°C for 2 h. Fractions
(approximately 1 ml each) were manually collected from the top of the gradient and RNA levels in the fractions were measured by a UV spectrophotometer at 260 nm (Nanodrop, Thermo Fischer Scientific). Aliquots of each
fraction were analyzed by immunoblotting or by ethidium bromide staining of ribosomal RNAs (rRNAs) purified using an ISOGEN LS kit (Nippon Gene).
5
Subcellular Fractionation of HT29-MTX Cells
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HT29-MTX cells were washed twice with ice-cold phosphate-buffered saline (PBS), scraped into 2 ml of 500 mM sodium carbonate (pH 11.0), transferred to a plastic tube, and homogenized with a Sonicator 250 apparatus (Branson Ultrasonic, Danbury, CT, USA) using three 20 s bursts. The homogenate was adjusted to 45% sucrose by the addition of 2 ml 90% sucrose prepared in 2 (N-morpholino) ethanesulfonic acid (MES)-buffered solution consisting of 25 mM MES-buffer solution (pH 6.5) and 0.15 M NaCl and placed at the bottom of an ultracentrifuge tube. A 5%–35% discontinuous sucrose gradient was formed (4 ml each of 5% and 35% sucrose, both in MES-buffer solution containing 250 mM sodium carbonate) and centrifuged at 40 000xg for 20 h in a Beckman SW41 Rotor (Beckman Coulter). Twelve fractions were collected and analyzed by 12% SDS-PAGE.
6
Caveolin-enriched Membrane Fraction Preparation
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INT-407 cells grown to confluence in 100-mm dishes were used to prepare caveolin-enriched membrane fractions as described previously.56 (link) Cells were washed twice with ice-cold PBS, scraped into 2 ml of 500 mM sodium carbonate (pH 11.0), transferred to a plastic tube, and homogenized with a Sonicator 250 apparatus (Branson Ultrasonic, Danbury, CT, USA) using three 20-s bursts. The homogenate was adjusted to 45% sucrose by the addition of 2 ml 90% sucrose prepared in 2-(N-morpholino) ethanesulfonic acid (MES)-buffered solution consisting of 25 mM MES-buffer solution (pH 6.5) and 0.15 M NaCl and placed at the bottom of an ultracentrifuge tube. A 5–35% discontinuous sucrose gradient was formed (4 ml each of 5% and 35% sucrose, both in MES-buffer solution containing 250 mM sodium carbonate) and centrifuged at 40 000 × g for 20 h in a Beckman SW41 Rotor (Beckman Coulter, Fullerton, CA, USA). Twelve fractions were collected and analyzed by 12% SDS-PAGE.
7
Caveolin-enriched membrane fractionation
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Cav-enriched membrane fractions were prepared as described previously.58 (link) Cells were washed twice with ice-cold PBS, scraped into 2 ml of 500 mM sodium carbonate (pH 11.0), transferred to a plastic tube, and homogenized with a Sonicator 250 apparatus (Branson Ultrasonic, Danbury, CT, USA) using three 20-s bursts. The homogenate was adjusted to 45% sucrose by the addition of 2 ml 90% sucrose prepared in 2-(N-morpholino) ethanesulfonic acid (MES)-buffered solution consisting of 25 mM MES-buffer solution (pH 6.5) and 0.15 M NaCl and placed at the bottom of an ultracentrifuge tube. A 5–35% discontinuous sucrose gradient was formed (4 ml each of 5 and 35% sucrose, both in MES-buffer solution containing 250 mM sodium carbonate) and centrifuged at 40 000 × g for 20 h in a Beckman SW41 Rotor (Beckman Coulter, Fullerton, CA, USA). Twelve fractions were collected and analyzed by 12% SDS-PAGE.
8
Subcellular Fractionation of Cells
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Cells were washed with cold PBS, scraped into 2 mL of 500 mM Na2CO3 (pH 11.0), and homogenized with a Sonicator 250 apparatus (Branson Ultrasonic, Danbury, CT). A discontinuous sucrose gradient (5, 35 and 45%) was formed and centrifuged at 40,000 × g for 20 h in a Beckman SW41 Rotor (Beckman Coulter, Fullerton, CA). Samples were divided to twelve fractions and analyzed by immunoblotting.
9
Subcellular Fractionation And Protein Analysis
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Cells were washed twice with ice-cold PBS, scraped into 2 mL of 500 mM sodium carbonate (pH 11.0), transferred to a plastic tube, and homogenized with a Sonicator 250 apparatus (Branson Ultrasonic, Danbury, CT) using three 20-sec bursts. The homogenate was adjusted to 45% sucrose by the addition of 2 ml 90% sucrose prepared in 2-(N-morpholino) ethanesulfonic acid (MES)-buffered solution consisting of 25 mM MES-buffer solution (pH 6.5) and 0.15 M NaCl and placed at the bottom of an ultracentrifuge tube. A 5–35% discontinuous sucrose gradient was formed (4 ml each of 5 and 35% sucrose, both in MES-buffer solution containing 250 mM sodium carbonate) and centrifuged at 40,000 × g for 20 h in a Beckman SW41 Rotor (Beckman Coulter, Fullerton, CA). Eight fractions were collected and analyzed by 12% SDS-PAGE.
10
Polysome Profiling of Metformin Treated Cells
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Briefly, metformin and control‐treated cells were grown to ~70% confluence. Cells were treated with 100 μg·mL−1 cycloheximide at 37 °C for 15 min prior to harvesting. Cells were lysed in lysis buffer (Pereboom et al., 2014 ), using homogenization on ice. Lysates were cleared by centrifugation at 1200 g for 10 min, and equal OD260 units were loaded onto a 17–50% sucrose gradient. Sucrose gradients were centrifuged for 2 h at 178 305 g in a Beckman SW41 rotor (Beckman Coulter, Indiana, USA) at 4 °C prior to fractionation. Fractionation was performed on an ISCO UV spectrophotometer and gradient fractionator (Teledyne ISCO, Nebraska, USA). Data were collected with labworks software (Lehi, UT, USA). Postcollection data analysis was performed in Microsoft Excel and graphpad prism 7 (La Jolla, CA, USA).
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