Cells (five 10-cm dishes) transfected with indicated plasmids were starved in EBSS for 2 hr and harvested as indicated above. Membranes from a 25,000×g membrane pellet were resuspended in 300 μl 60% (wt/vol) Nycodenz (Accurate Chemical, Westbury, NY) in B88 buffer and transferred to a Beckman tube (Polycarbonate, 11 × 34 mm). Aliquots were overlaid with 600 μl of 40% Nycodenz in B88 buffer and 100 μl B88 buffer, and then centrifuged for 2 hr at 100,000×g (TLS 55 rotor, Beckman). Ten fractions were collected from top to bottom and analyzed by immunoblot. For determining the level of IL-1β in the membrane fraction, top fractions were combined and diluted with B88 buffer and membranes were collected by centrifugation at 100,000×g for 40 min followed by immunoblot analysis.
Nycodenz
Manufactured by Beckman Coulter
Sourced in United States
Nycodenz is a non-ionic water-soluble iodinated density gradient medium used for the separation and isolation of cells, organelles, and other biological particles by density gradient centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Tls 55 rotor, by Beckman Coulter (2 mentions)
Sw41 tube, by Beckman Coulter (2 mentions)
Nycodenz, by Progen Biotechnik (2 mentions)
Ultra clear, by Beckman Coulter (2 mentions)
Sw40ti rotor, by Beckman Coulter (2 mentions)
Nycodenz, by Merck Group (1 mentions)
Ultra clear centrifuge tube, by Beckman Coulter (1 mentions)
Sw41 rotor, by Beckman Coulter (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
7 protocols using nycodenz
1
Subcellular Fractionation and IL-1β Quantification
2
Subcellular Fractionation of Cell Lysates
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Subcellular fractionation was performed as described previously. In brief, cells were cultured in the presence or absence of 50 μg mL–1 of β-cycloheximide for 5 h and then scraped into media with the use of a disposable cell scraper. Cells were spun down at 4 °C and 500× g for 5 min and washed sequentially with cold PBS and ice-cold homogenization buffer (25 mM Tris/Cl pH7.5 and 130 mM KCl). Cell pellets were resuspended in homogenization buffer containing protease inhibitors and homogenized by passing through a 25-gauge needle 20 times on ice. Lysates were cleared by a centrifugation at 4 °C and 1000× g for 5 min. Post-nuclear supernatants were overlaid on a discontinuous Nycodenz gradient pre-made in an SW41 tube (Beckman Coulter, Indianapolis, IN, USA) as 0.66 mL of 40% and 5 mL each of 25% and 5% Nycodenz (Axis-Shield/Alere Technologies AS, Oslo, Norway). After gradients were centrifuged at 4 °C and 30,000 rpm for 1 h, 13 fractions at 1 mL each, were collected from top to bottom of each gradient. Equal volumes of each fraction were used for precipitating proteins with 3 volumes of methanol/chloroform (2:1, v/v). After being dried at 37 °C for 1 h, precipitated proteins in each fraction were used for Western blot.
3
Lysosome Enrichment from Mouse Livers
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For each sample and replicate, a pool of three mouse livers was centrifuged in a discontinuous Nycodenz (Progen Biotechnik) density gradient as previously described33 (link), with modifications. Briefly, tissues were homogenized in an assay buffer (0.25 M sucrose, pH 7.2) and centrifuged first at 4,800 X g for 5 min, and then at 17,000 X g for 10 min. The sediment of the second centrifugation was washed at 17,000 X g for 10 min, resuspended 1:1 vol/vol in 84.5% Nycodenz, and placed on the bottom of an Ultraclear (Beckman) tube. On top, a discontinuous gradient of Nycodenz was constructed (layers from bottom to top were: 32.8%, 26.3%, and 19.8% Nycodenz). Samples were then centrifuged for 1 h in an SW 40 Ti rotor (Beckman) at 141,000 X g. Lysosome-enriched fractions were collected from the 26.3/19.8 interface, diluted in 5–10 volumes of assay buffer, and centrifuged at 37000 X g for 15 min. Pellets were resuspended in 500 μl of assay buffer.
4
Subcellular Fractionation of Cells
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Cells (1 × 108) were washed with PBS and resuspended in 1 ml cold buffer A (250 mM, 10mM HEPES, pH 7.4, 1 mM EDTA) supplemented with 1mM PMSF and protease inhibitor cocktail (Roche). Cells were disrupted by repeated aspiration through 26 gauge needle and centrifuged (2,000 rpm at 4°C for 10 min) and 0.95 ml supernatant was diluted with 1.45 ml 85.6% Nycodenz (Sigma) solution to become 52%, then placed on the bottom of the 13 ml ultra-clear centrifuge tube (Beckman). A discontinuous Nycodenz gradient (26%, 24 %, 20%, 15%) was layered on the top of the 52% supernatant and centrifuged at 24,700 rpm at 4°C for 3 h in a SW41 rotor (Beckman). Fractions were washed with cold PBS and collected as indicated (A1, A2, L, M). Pellets were collected at 14,800 rpm, 4°C for 1 h and used for immunoblotting experiments.
5
Lysosome Enrichment from Mouse Livers
6
Subcellular Distribution of XK-EGFP Reporter
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To examine whether the XK-EGFP reporter mimicked endogenous XK in subcellular distribution, STHdhQ7/Q7 cells in 10-cm dishes were transfected with pcDNA3.1-XK-EGFP for 16 h and cultured further in the presence or absence of 50 μg/ml of β-cycloheximide for 5 h. Cells were then scraped into culture media and collected by centrifugation at 4°C 1,000 rpm for 5 min. After sequential washes with cold PBS and cold homogenization buffer (25 mM Tris/Cl pH 7.5 and 130 mM KCl), cells were resuspended in homogenization buffer containing protease inhibitors and homogenized by passing through a 25-gauge needle 20 times on ice. Lysates were cleared by centrifugation at 4°C and 2,000 rpm for 5 min. Postnuclear supernatants were overlaid on a discontinuous Nycodenz gradient premade in an SW41 tube (Beckman Coulter) as 0.66 ml of 40% and 5 ml each of 25 and 5% Nycodenz (Axis-Shield/Alere Technologies AS) and centrifuged at 4°C and 30,000 rpm for 1 h. Twelve fractions of 1 ml each were collected from top to bottom of each gradient. Equal volumes of each fraction were used for precipitating the proteins with three volumes of methanol/chloroform (2:1, v/v). After being dried at 37°C for 1 h, precipitated proteins in each fraction were resuspended in 1XSDS sample buffer and analyzed by SDS-PAGE and Western blot.
7
Density Gradient Fractionation of Adipocyte Membranes
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Adipocytes were incubated in the presence of FSK and CQ, and total membrane fractions were collected as described in the previous paragraph. Total membranes were suspended in 0.3 ml B88 buffer containing 60% (wt/vol) Nycodenz (Accurate Chemical) and overlaid with 0.6 ml B88 containing 40% Nycodenz and 0.1 ml B88. The Nycodenz gradient was centrifuged at 250,000 g for 90 min (TLS 55 rotor; Beckman), and subsequently 10 fractions, 0.1 ml each, were collected from the top and analyzed by immunoblot for the indicated markers.
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