A discontinuous iodixanol gradient (40% (w/v), 20% (w/v), 10% (w/v) and 5% (w/v) solutions of iodixanol) was prepared by diluting a 60% OptiPrepTM stock (Axis-Shield PoC, Norway) with appropriate amounts of PBS. The gradient was formed by layering 2 mL of 40%, 2 mL of 20%, 2 mL of 10% and 2 mL of 5% solutions on top of each other in a 14 × 95 mm polyallomer tube (Beckman Coulter). Concentrated conditioned medium (CCM) was harvested by successive centrifugations (10 min 750 × g, 10 min, 25°C; 2,000 × g, 20 min, 4°C; 10,000 × g, 30 min, 4°C; filtered through a 0.22 μm membrane; 10,0000 × g, 90 min, 4°C). 1 mL CCM sample was overlaid onto the top of the gradient which was then centrifuged for 18 h at 100,000 g and 4°C (SW 40 Ti rotor, Beckman Coulter). Resulting fractions were determined by visual inspection and removed sequentially from the top using a pipette. Each fraction was diluted to 50 mL in PBS to remove OptiPrepTM, and the resulting pellets were resuspended in 100 μL PBS and stored at −80°C. The fraction at the layer between 40% and 20% density from the OptiPrepTM density gradient was used for the experiments.
Polyallomer tube
Manufactured by Beckman Coulter
Sourced in United States
Polyallomer tubes are a type of laboratory equipment used for a variety of applications, including sample storage, centrifugation, and other scientific procedures. They are made from a durable, chemically resistant material that is compatible with a wide range of solvents and reagents. Polyallomer tubes are designed to provide reliable and consistent performance in a laboratory setting.
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Lab products found in correlation
Sw40 rotor, by Beckman Coulter (3 mentions)
Sw40ti rotor, by Beckman Coulter (2 mentions)
Model 11, by Brandel Inc (2 mentions)
Sw60ti rotor, by Beckman Coulter (2 mentions)
Ultracentrifuge, by Beckman Coulter (2 mentions)
Gradient master, by Beckman Coulter (2 mentions)
P28s rotor, by Hitachi (2 mentions)
Dc protein assay kit, by Bio-Rad (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
0.22 μm filter, by Merck Group (2 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Steriflip filter unit, by Merck Group (1 mentions)
Sw41 rotor, by Beckman Coulter (1 mentions)
L7 55 centrifuge, by Beckman Coulter (1 mentions)
Phosphate buffered saline (pbs), by Dutscher (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Gradient master, by BioComp Instruments (1 mentions)
0.2 m filter, by Merck Group (1 mentions)
Qiazol, by Qiagen (1 mentions)
33 protocols using polyallomer tube
1
Iodixanol Density Gradient Purification of Extracellular Vesicles
2
Polysome Profiling: Dissecting Translational Dynamics
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We followed the polysome profiling protocol described in Esposito et al. (2010) (link) with some minor modifications. Specifically, about 20% of the supernatant was saved for cytoplasmic RNA extraction. The rest of the supernatant was transferred to a new tube and was centrifuged at 14,000 x g for 5 min to remove mitochondria. The resulting supernatant was layered onto a 10 mL 10%–50% linear sucrose gradient in a polyallomer tube (Beckman Coulter), containing 20 mM HEPES-KOH pH 7.5, 15 mM MgCl2, 80 mM KCl, 2mM DTT, and 100 μg/ml cycloheximide, followed by centrifugation in an SW-41 Ti rotor at 39,000 rpm at 4°C for 2 hr. The gradient was then fractionated using a system comprising a syringe pump (Harvard Apparatus model 11), a density gradient fractionator (Brandel), and an ISCO UA-6 UV/VIS detector. The lysate was partitioned into three fractions based on UV absorbance, i.e., ribosome-free, monosome, and polysome fractions. Monosome and polysome fractions were incubated at 65°C for 5 min with 25 mM EDTA (pH 8.0), 10 mM Tris-HCl (pH7.0), and 1% SDS, followed by extraction of RNA with phenol-chloroform and Ethanol precipitation.
3
Sucrose Density Gradient Separation of IgE-Sensitized BMMCs
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Sucrose density gradient separations were performed as previously described (42 (link)). Briefly, IgE-sensitized BMMCs isolated from WT or ORMDL2,3 dKO mice were used. BMMCs (15 × 106) were then activated or not with antigen (TNP-BSA; 0.25 μg/ml) for 5 min. After centrifugation the cells were lysed on ice in 0.8 ml lysis buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 2 mM EDTA, 10 mM glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, protease inhibitor cocktail) supplemented with 1% Brij-96. The gradient was formed by adding 0.5 ml of 80% sucrose stock solution to the bottom of the polyallomer tube (13 × 51 mm; Beckman Instruments) followed by 1.5 ml of 40% sucrose containing the cell lysate, 2 ml of 30% sucrose, and 1 ml of 10% sucrose. After 4 h centrifugation at 4°C and 210,000g, using an SW55 Ti rotor, nine fractions were collected from the top of the gradient, and individual fractions were size-fractionated by SDS-PAGE followed by immunoblotting with PY20-HRP conjugate or LYN-specific antibody. The position of phosphorylated PAG, LYN, LAT1, and LAT2 is known from our previous studies (43 (link), 44 (link)).
4
Polysome Profiling: Dissecting Translational Dynamics
5
Virus Purification and Extraction Protocol
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To harvest virus, supernatants were clarified by low-speed centrifugation, filtered through a 0.45-μm cellulose acetate syringe filter (catalog number 431220; Corning), and ultracentrifuged in a polyallomer tube (catalog number 331372; Beckman) at 35,000 rpm for 45 min at 4°C. The supernatant was removed, and the virus pellet was resuspended in 1 ml of phosphate-buffered saline (PBS). The sample was incubated at room temperature for 15 min and brought up to 4 ml with PBS. The virus pellet was then subjected to a step gradient centrifugation using a 20% sucrose cushion at 35,000 rpm for 2 h at 4°C. The supernatant was removed and the pellet resuspended in 1 ml of lysis buffer (0.1 mM EDTA, 150 mM NaCl, 5 mM MgCl2, 5 mM CaCl2, 1 mM dithiothreitol [DTT], 20 mM HEPES-KOH, pH 7.9, 1% Triton X-100, protease inhibitor tablet [1 per 10 ml]). Meanwhile, the intracellular proteins were collected by washing cells once with PBS and resuspending them in 1 ml of lysis buffer.
6
Sucrose Gradient Centrifugation for Amyloid Fibrils
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For sucrose‐gradient centrifugation, solutions containing 50 mm Tris (pH 7.4), 0.1% NP‐40 and sucrose (10%, 20%, 30%, 40%, 50% and 60%) were pipetted into a 4‐mL 11 × 60 mm polyallomer tube (Beckman Coulter, Brea, CA, USA) starting with 200 µL of 60% sucrose solution loaded to the bottom, followed by 400 µL layers of 50–10% sucrose solution. In vitro‐formed fibrils [200 µL at a concentration of 5 µm (monomer equivalent)] were loaded on the top of the sucrose gradient. Ultracentrifugation was performed with 40 000 r.p.m. (163 900 g ) at 4 °C for 1 h using a Sw60Ti rotor (Beckman Coulter). Samples were harvested after centrifugation from the top to the bottom of the sucrose gradient in 200‐µL fractions (i.e. fraction 1 represents the top of the gradient, fraction 12 the bottom fraction). 28 µL of each fraction was used for western blot analysis. aSyn was detected using an antibody against full‐length aSyn (4B12; BioLegend, San Diego, CA, USA).
7
Production of Pseudotyped Virus Stocks
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Virus stocks were generated by transient transfection of HEK293T cells using polyethylenimine (PolySciences). Virus stocks were produced with env-deleted molecular clones and pseudotyped with the VSV-G protein or HIV-1 Env of the YU2 strain by cotransfection with expression vectors for the respective envelope proteins. The Gag-Pol expression vector pCRV1-Gag-Pol along with the pHCMV-G plasmid were used to cotransfect with shRNA-encoding lentivirus vectors. For producing high-titer virus stocks, HEK293T cells were seeded in 100-mm plates at 5 × 106 cells per plate. Culture medium was left untouched or replaced with fresh medium 1 day after transfection. Supernatant harvested at 2 days posttransfection was filtrated with a Steriflip filter unit (0.45 μm, polyvinylidene difluoride [PVDF]; Millipore) and gently layered onto 5 mL of 20% (weight per volume) sucrose solution in a polyallomer tube (Beckman Coulter). Ultracentrifugation was performed for 2 h at 24,000 rpm and 4°C using an SW28 rotor. Virus pellets were incubated with 3.2 mL of culture medium for at least 1 h at 4°C before suspension. Aliquots were prepared and stored at −80°C until use.
8
Sucrose Gradient Separation of IgE-Activated Mast Cells
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Sucrose density gradient separations were performed as previously described (101 (link)). Briefly, IgE-sensitized BMMCs (15 × 106) were pretreated or not with UA for 15 min and then activated or not with antigen (250 ng/ml) for 5 min. After centrifugation, the cells were lysed on ice in 0.8 ml lysis buffer (10 mM Tris–HCl, pH 8.0, 50 mM NaCl, 2 mM EDTA, 10 mM glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, 0.5 U/ml aprotinin, and 0.5 U/ml leupeptin) supplemented with 1% Brij-96. The gradient was formed by adding 0.5 ml of 80% sucrose stock solution to the bottom of a polyallomer tube (13 × 51 mm; Beckman Instruments) followed by 1.5 ml of 40% sucrose containing the cell lysate, 2 ml 30%, and 1 ml 5% sucrose. After 4 h centrifugation at 4 °C (210,000g using an SW55 Ti rotor), 10 fractions were collected from the top of the gradient, and individual fractions were analyzed by SDS-PAGE followed by immunoblotting with PY-20-HRP and protein-specific antibody.
9
Isolation and Analysis of TG-rich Lipoproteins
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The human plasma for research was purchased from Korean Red Cross (Busan, Republic of Korea). To obtain the TG-rich lipoprotein from the human or murine serum, we performed TG-rich lipoprotein isolation following pervious study [36] . We allocated serum to the polyallomer tube (Beckman Coulter, Pasadena, CA) and placed same volume of NaCl (d=1.006g/ml) solution on the serum. The tube was centrifuged by ultracentrifuge (Beckman) with SW 40 rotor (Beckman) by 40, 000 rpm for 24 hour at 4 ºC. After centrifugation, the upper 10% of total volume was obtained and the TG contents were measured by TG assay kit as described above. In addition, the TG-rich lipoprotein sample was mixed with SDS sample buffer and loaded into SDS-PAGE for silver staining. For the experiments to assess the effect of TG-rich lipoprotein in in vitro model, TG-rich lipoprotein was treated with TG concentration of 0.2 mg/ml for 24 hours and the TG accumulation was observed through ORO staining or TG assay.
10
Sucrose Gradient Fractionation of Yeast Polysomes
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Yeast cells were grown exponentially at 30 °C and treated 0.1 mg/ml of cycloheximide for 5 min before harvesting, then harvested by centrifugation. The harvested cell pellet was frozen and ground in liquid nitrogen using a mortal. The cell powder was resuspended with lysis buffer (20 mM HEPES-KOH, pH 7.4, 100 mM potassium acetate, 2 mM magnesium acetate) to prepare the crude extracts. Sucrose gradients (10–50% sucrose in 10 mM Tris-acetate, pH 7.4, 70 mM ammonium acetate, and 4 mM magnesium acetate) were prepared in 25 × 89 mm polyallomer tubes (Beckman Coulter) using a Gradient Master. Crude extracts (the equivalent of 50 A260 units) were layered on top of the sucrose gradients and then centrifuged at 150,000×g in a P28S rotor (Hitachi Koki, Japan) for 2.5 h at 4 °C. The gradients were then fractionated (TOWA Lab, Tsukuba). The polysome profiles were generated by continuous absorbance measurement at 254 nm using a single path UV-1 optical unit (ATTO Biomini UV-monitor) connected to a chart recorder (ATTO digital mini-recorder). Equal volumes of the fractions were collected and processed for western blotting.
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