For density gradient fractionation, the lysate was layered on a sucrose gradient (10%–50%) and ultracentrifugation was performed using a Beckman SW-40Ti rotor and Beckman Optima L-100 XP ultracentrifuge, with a speed of 35,000 rpm at 4°C for 160 min. Fractionations and UV absorbance profiling were carried out using Density Gradient Fractionation System (Teledyne ISCO). For sucrose cushioning, the lysate was layered on a 20% sucrose solution (20% sucrose, 10 mM Tris-HCl pH 7.4, 300 mM KCl,10 mM MgCl2), which contains a high concentration of KCl to avoid the aspecific binding of proteins to ribosomes. Then, ultracentrifugation was performed using a Beckman SW-55Ti rotor and Beckman Optima L-100 XP ultracentrifuge, with a speed of 41,000rpm at 4°C for 120 min. Proteins were precipitated from each fraction using methanol-chloroform precipitation and pellets were resuspended in 1x NuPAGE LDS sample buffer and used for western blotting as described below. RNA from each fraction was isolated as described below.
Beckman sw55ti rotor
Manufactured by Beckman Coulter
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The Beckman SW55Ti rotor is a high-speed ultracentrifuge rotor designed for use in Beckman Coulter ultracentrifuge systems. It is capable of reaching speeds up to 55,000 rpm and can achieve relative centrifugal forces up to 347,000 x g. The rotor is compatible with a variety of sample tube sizes and can be used for a range of applications that require high-speed centrifugation.
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5 protocols using beckman sw55ti rotor
1
Sucrose Density Gradient Fractionation
2
Microsome Subfractionation in Arabidopsis
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Microsome subfractionation was performed according to Ishikawa et al. (2005) (link). Total microsomes were prepared as follows: A. thaliana leaf tissue was homogenized in a buffer containing 50mM TRIS-acetate (pH 7.5), 250mM sorbitol, 2mM EGTA, 2mM MgCl2, 2mM DTT supplemented with Complete protease inhibitor cocktail. The homogenate was filtered and centrifuged at 10 000 g for 10min at 4 °C. The supernatant (S10) was further centrifuged at 100 000 g (rav) in a Beckman SW55Ti rotor (Beckman Instruments) for 2h at 4 °C. The resulting pellet (P100), containing total microsomes, was resuspended in 10mM Tricine-KOH (pH 7.5), 1mM EGTA, 2mM MgCl2, 5% (w/w) sucrose, and was loaded on a sucrose density gradient [10.4ml, 15–45% (w/w) sucrose in 10mM Tricine-KOH (pH 7.5), 1mM EGTA, and 2mM MgCl2] and centrifuged at 77 000 g (rav) for 19h in a Beckman SW40 rotor. Twenty fractions of 0.55ml were collected. The precipitate at the bottom of the tube was solubilized with 550 μl of SDS–PAGE loading buffer. Equal volumes of each fraction and of the solubilized precipitate were analyzed by SDS–PAGE and protein blot.
3
Purification of Rotavirus Double-Layered Particles
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MA104-cell monolayers in 10 cm cell culture plates were infected with rSA11 viruses at an MOI of 5 and harvested at 12 h p.i. Cells were lysed by adjusting media to 0.5% Triton X100 (Sigma) and incubation on ice for 5 min. Lysates were then clarified by centrifugation at 500× g at 4 °C for 6 min. The clarified lysates were adjusted to 10 mM EDTA and incubated for 1 h at 37 °C to cause the conversion of rotavirus TLPs to DLPs [45 (link)]. CsCl was added to samples to a density of 1.367 g/cm3 and samples were centrifuged at 110,000× g with a Beckman SW55Ti rotor (Beckman Coulter Life Sciences, Indianapolis, IN, USA) at 8 °C for 22 h. Fractions containing viral bands were recovered using a micropipettor and fraction densities were determined using a refractometer.
4
Subcellular Fractionation of SH-SY5Y Cells
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SH-SY5Y-APP/BACE1 cells were washed twice with ice-cold PBS and harvested with 0.25% trypsin-EDTA. Cells were then lysed in detergent-free condition using morpholinoethanesulfonic acid-buffered saline (MBS; 25 mM MES, 150 mM NaCl, pH 6.5) containing 500 mM sodium carbonate (Sigma, #S7795) and a protease inhibitor cocktail (Millipore, #535140). Cell lysates were homogenized 20 times with a 2 mL homogenizer and 10 times through a 22-gauge needle. Next, cell lysates were sonicated for 1 min (20 s sonication followed by 10 s intervals). Equal amounts of proteins were added to 0.8 mL of 80% (w/v) sucrose in MBS. Then, 1.6 mL of 35% (w/v) sucrose and 5% (w/v) sucrose in MBS were placed in a 5.1 mL ultracentrifuge tube (Beckman Coulter, #326819) to form a discontinuous sucrose gradient. These tubes were placed in a Beckman SW 55Ti rotor (Beckman Coulter) and centrifuged at 50,000 rpm for 3 h at 4 °C. From the top to the bottom, 12 fractions (0.4 mL each) were collected.
5
Dynactin Disruption Assay using Purified Proteins
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For disruption experiments using purified proteins, 10 μg (9 × 10−6 mole) of purified bovine dynactin was mixed with a 25× molar excess (9 × 10−4 mole; see Maier et al., 2008 (link)) of the different recombinant dynamitin constructs in a total volume of 0.1–0.2 ml of sedimentation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM dithiothreitol [DTT]) and then incubated on ice for 30 min. In some cases, 9 × 10−4 mole of the purified recombinant dynamitin fragments was added to 500 μg of Cos-7 detergent lysates (prepared as described later). After incubation, the samples were sedimented into a 5–20% sucrose gradient in a Beckman SW55 Ti rotor (Beckman Coulter, Brea, CA) at 30,500 rpm for 16 h at 4°C. Fractions (0.5 ml) were collected, and dynactin subunits were detected using SDS–PAGE, followed by immunoblotting. For analysis of dynactin integrity in cells overexpressing different dynamitin constructs, cells were harvested 18–48 h posttransfection, lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Triton X-100) for 30 min at 4°C, and then centrifuged at 44,700 rpm in a Beckman SW55Ti rotor for 30 min. The supernatants were collected, and 500 μg to 1 mg of protein in a volume of 0.2 ml was sedimented into a 5–20% sucrose gradient and analyzed as described.
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