Swc579–303 was mixed with 147 bp Widom 601 sequence-containing nucleosomes at a 2.5:1 molar ratio in H50 buffer (10 mM HEPES pH 7.5, 50 mM NaCl, 1 mM DTT(Dithiothreitol)). Precipitated material was removed by centrifugation at 16,000 × g for 10 min. The sample was chemically cross-linked using the GraFix method in a 10–30% glycerol gradient (Stark, 2010 (link)), prepared with a Gradient Master instrument (Biocomp) from top (H50 + 10% vol/vol glycerol) and bottom (H50 + 30% vol/vol glycerol, 0.15% vol/vol glutaraldehyde) solutions. The sample was applied on top of the gradient and centrifuged in a SW40Ti rotor (Beckman-Coulter) at 35,000 rpm, 4°C for 16 hr. Fractions were collected manually by pipetting from the top, and the reaction was quenched by adding Tris pH 7.5 to each fraction to 50 mM. Fractions were analyzed using a 6% native gel (29:1 Acrylamide/Bis) stained with ethidium bromide and negative-stain EM with uranyl formate to select for monodisperse particles of the complex. Selected fractions were dialyzed into H50 buffer supplemented with 0.1 mM PMSF and concentrated to ~1 mg/ml.
Gradient master instrument
Manufactured by BioComp Instruments
The Gradient Master instrument is a precision laboratory device designed for the preparation of linear and step gradients. It enables the creation of gradients by precisely controlling the mixing of liquids. The Gradient Master is a versatile tool for a range of scientific applications that require the generation of controlled gradient solutions.
Automatically generated - may contain errors
Lab products found in correlation
Sw40ti rotor, by Beckman Coulter (2 mentions)
2110 fraction collector, by Bio-Rad (1 mentions)
Optima l 90k, by Beckman Coulter (1 mentions)
Piston gradient fractionator, by Bio-Rad (1 mentions)
Superase in, by Thermo Fisher Scientific (1 mentions)
Sw41ti rotor, by Beckman Coulter (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
4 protocols using gradient master instrument
1
Structural Characterization of Swc5-Nucleosome Complex
2
Sucrose Gradient Fractionation of Cellular Components
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A total of 10 to 40% sucrose gradients were prepared (solvated in 25 mM HEPES Tris, 100 mM potassium glutamate, 20 mM magnesium acetate, 14 mM β-mercaptoethanol, and 0.5% Tween 20) in polyclear centrifuge tubes (Seton Scientific) using a Gradient Master instrument (BioComp Instruments) the day of culture harvest and kept at 4°C until lysates were prepared. A total 200 μL of normalized lysates were added to the tops of the gradients. Cellular components were separated in the gradients using a Beckman Coulter Optima L-90K ultracentrifuge with a SW41 Ti rotor at 35,000 RPM for 3.5 h at 2°C. The gradients were then fractionated using a BioComp Piston Gradient Fractionator with a Bio-Rad 2110 Fraction Collector while absorbance was measured at 258 nm.
3
Nucleosome-Swc5 Complex Formation
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Swc579–303 was mixed with 147 bp Widom 601 sequence-containing nucleosomes at a 2.5:1 molar ratio in H50 buffer (10 mM HEPES pH 7.5, 50 mM NaCl, 1 mM DTT). Precipitated material was removed by centrifugation at 16,000 × g for 10 min. The sample was chemically cross-linked using the GraFix method in a 10–30% glycerol gradient55 (link), prepared with a Gradient Master instrument (Biocomp) from top (H50 + 10% v/v glycerol) and bottom (H50 + 30% v/v glycerol, 0.15% v/v glutaraldehyde) solutions. The sample was applied on top of the gradient and centrifuged in a SW40Ti rotor (Beckman-Coulter) at 35,000 rpm, 4 °C for 16 h. Fractions were collected manually by pipetting from the top, and the reaction was quenched by adding Tris pH 7.5 to each fraction to 50 mM. Fractions were analyzed using a 6% native gel stained with ethidium bromide and negative-stain EM with uranyl formate to select for monodisperse particles of the complex. Selected fractions were dialyzed into H50 buffer supplemented with 0.1 mM PMSF and concentrated to ~1 mg/ml.
4
Polysome Profiling of Mouse Gastrocnemius Muscle
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One gastrocnemius muscle from each mouse was pulverized under cryogenic conditions in a cryo‐freezer grinder (SpexSamplePrep, 10 cps 3 × 2 min) in 2.5 mL of lysis buffer (20 mM Tris‐HCl, pH = 7, 100 mM NaCl, 50 mM NH4Cl, 10 mM MgCl2, 1% Triton X‐100) and freshly added 100 μg/mL cycloheximide, 1 mM dithiothreitol supplemented with a protease inhibitor cocktail tablet (Roche), and 200 U of SUPERase*In (Invitrogen). After 2 h incubation at 4°C on a rotating wheel, lysates were disrupted by five up‐and‐down passages through a 19‐gauge syringe and clarified by centrifugation for 10 min at 12 000 rcf at 4°C. Polysome profiling was further carried out as previously described by Ingolia and colleagues.28 Briefly, equal loading was assured by measuring OD260 for each sample. Lysates were then layered on top of a pre‐cooled 50–10% sucrose gradient (dissolved in 8.3 mM Tris‐HCl, pH = 7.5, 8.3 mM NH4Cl, 2 mM MgCl2 freshly supplemented with 0.083 mM dithiothreitol, 100 μg/mL cycloheximide, and 200 U of SUPERase*In). Gradient was formed using a Gradient Master instrument (Biocomp) according to the manufacturer's instruction. Samples were centrifuged at 150 000 rcf for 3 h at 4°C using a Beckman SW41Ti rotor. Profiles of the different samples were obtained at a speed of 0.5 mL/min and continuous measurement of the absorbance at 254 nm.
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