Gradient master

Manufactured by Beckman Coulter

The Gradient Master is a laboratory instrument used to create linear gradients. It precisely controls the mixing of two solutions to generate a continuous gradient. The device can be used in various applications, such as density gradient ultracentrifugation, where it is essential for separating and purifying biological samples.

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Lab products found in correlation

Polyallomer tube, by Beckman Coulter (2 mentions) P28s rotor, by Hitachi (2 mentions) Thinwall polypropylene tube, by Beckman Coulter (1 mentions) Hiprep 26 60 sephacryl s 200 high resolution column, by GE Healthcare (1 mentions) Optima xl 100k, by Beckman Coulter (1 mentions) Hiprep, by Cytiva (1 mentions) Superase in rnase inhibitor, by Thermo Fisher Scientific (1 mentions) Sw41ti rotor, by Beckman Coulter (1 mentions)

4 protocols using gradient master

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.

Matsuo Y., Ikeuchi K., Saeki Y., Iwasaki S., Schmidt C., Udagawa T., Sato F., Tsuchiya H., Becker T., Tanaka K., Ingolia N.T., Beckmann R, & Inada T. (2017). Ubiquitination of stalled ribosome triggers ribosome-associated quality control. Nature Communications, 8, 159.

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Ferritin Monomer Sedimentation Profiling

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5–30% (w/v) sucrose gradients in PBS were prepared in Beckman Coulter thin wall polypropylene tubes (14 mL) using a BioComp Gradient Master automatic gradient maker. Ferritin monomer samples were layered on top of the gradient and resolved on a Beckman Coulter Optima XL-100K in an SW40-Ti rotor at 38,000 rpm for 2.5 hours at 4°C. Twenty four 0.5 mL fractions were manually collected from the top of the gradient in a top-down manner such that the early fractions represent the top of the gradient. Fractionated samples were buffer exchanged by size exclusion chromatography to remove the sucrose. Note that fractionated samples of Sigma-Aldrich ferritin were buffer exchanged on a HiPrep 26/60 Sephacryl S200 high resolution column (GE Healthcare). Fraction 17 was the furthest fraction, for both the Sigma-Aldrich and Amersham preparations, that provided sufficient material for subsequent analyses.

Ghirlando R., Mutskova R, & Schwartz C. (2015). Enrichment and Characterization of Ferritin for Nanomaterial Applications. Nanotechnology, 27(4), 045102.

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Polysome Profiling in Yeast Cells

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Yeast cells were grown exponentially at 30 °C and harvested by centrifugation. Cell extracts were prepared as described previously20 (link). The equivalent of 50 A260 units were layered onto linear 10–50% sucrose density gradients. 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 polyallomer tubes (Beckman Coulter) using a Gradient Master. Crude extracts were layered on top of the sucrose gradients and centrifuged at 150,000 × g in a P28S rotor (Hitachi Koki, Japan) for 3 hours at 4 °C. Gradients were then fractionated (TOWA lab, Tsukuba). 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 volume fractions were collected and processed for Northern or Western blotting as described above37 (link).

Ikeuchi K, & Inada T. (2016). Ribosome-associated Asc1/RACK1 is required for endonucleolytic cleavage induced by stalled ribosome at the 3′ end of nonstop mRNA. Scientific Reports, 6, 28234.

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Polysome Profiling of Brain Tissue

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Whole brains were extracted, immediately frozen in liquid nitrogen, and stored at −80°C. Brains were pulverized under liquid nitrogen using a previously cooled, RNAse free mortar and pestle. The powder obtained was transferred to a 10-cm plate on dry ice while subsequent brains were pulverized. Afterward, 1 mL of lysis buffer (10 mM Tris-HCl at pH 8.0; 150 mM NaCl; 5 mM MgCl₂; 1 mg/mL heparin; 1% Nonidet-P40; 0.5% deoxycholate; 40 mM DTT; 1 U/mL SUPERaseIn RNAse inhibitor [Thermo Fisher]; and 150 μg/mL cycloheximide) was added to the tissue powder. Next, re-suspended powder was scraped from the plate and transferred to a microcentrifuge tube with pipetting 10 times to lyse the cells. The cell nuclei and cell debris were removed by centrifugation (2,000g, 10 min, at 4°C). The supernatant was transferred to a fresh tube and then centrifuged again at 16,000g for 7.5 minutes at 4°C. 400 μL of supernatant was layered onto a 10 mL linear sucrose gradient (15-45% sucrose [w/v] made using a Biocomp Gradient Master) and centrifuged in a SW41Ti rotor (Beckman) for 125 minutes at 38,000 rpm at 4°C with the brake off. Polysome profiles were recorded using a UA-6 absorbance (ISCO) detector at 254 nm and fractions were collected along the gradient. Fractions were used for subsequent western blot analysis.

Eagleman D.E., Zhu J., Liu D.C., Seimetz J., Kalsotra A, & Tsai N.P. (2020). Unbiased Proteomic Screening Identifies a Novel Role for the E3 Ubiquitin Ligase Nedd4-2 in Translational Suppression During ER Stress. Journal of neurochemistry, 157(6), 1809-1820.

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