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Ultra turrax homogeniser

Manufactured by IKA Group
Sourced in Germany

The Ultra-Turrax is a high-speed homogenizer designed for the efficient dispersion and emulsification of liquids, pastes, and slurries. It features a powerful motor and a rotor-stator system that generates high shear forces to break down and blend various materials into a homogeneous mixture.

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5 protocols using ultra turrax homogeniser

1

Isolation of Membrane Protein Fractions

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Transfected 293T cells were incubated with PBS containing 0.04% EDTA (pH 7.4) at 37 °C for 10 min. Cells were spun at 1000g for 10 min and the pellet was suspended in 3 ml of ice-cold Tris-HCl (5 mM), pH 7.4. Pellets were homogenised (Ultra-Turrax homogeniser; IKA Werke, Staufen, Germany) and centrifuged at 48 000g at 4 °C for 15 min. The obtained pellet was re-suspended in 10 ml Tris-HCl (50 mM; pH 7.4) and centrifuged at 48 000g for 15 min at 4 °C. The pellet was resuspended in 10 ml of Tris-HCl (50 mM; pH 7.4) and spun at 48 000g for 15 min, at 4 °C. The resulting pellet was resuspended in 6 ml Tris-HCl (50 mM; pH 7.4) and total protein concentration was determined using the BCA protein assay (Pierce Biotechnology Inc., Rockford, IL) according to the manufacturer's protocol.
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2

Metabolite Extraction from Milk and Ricotta

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The extraction of metabolites from both milk creams and ricotta samples was carried out as previously reported [19 (link),20 (link)], with some modifications. Briefly, 2 g of each sample was extracted using a 10-millilitre mixture containing 80:20 (v/v) methanol:water, added with 0.1% of formic acid, with an Ultra-Turrax homogeniser (IKA-Werke, Staufen im Breisgau, Germany) for 4 min at room temperature. Next, the samples were centrifuged at 12,000× g for 10 min at 4 °C and the supernatants were incubated overnight in a freezer (−18 °C) following the addition of a 5% TCA solution, to remove large biomolecules (such as proteins). The supernatants were then filtered through 0.2-micrometre cellulose membranes and transferred to amber vials for the further metabolomic analysis. Each sample was analysed considering three biological replications.
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3

Colonic Citrate Synthase Activity Assay

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Frozen colonic tissues were homogenised on ice with an ultra-turrax homogeniser (IKA-Werke GmbH & Co., Germany) in a cold buffer (composition: sucrose 250 mM, Tris 5 mM, EGTA 1 mM, Triton X-100 0.02%, pH 7.4). Then, homogenates were centrifuged at 12,000× g for 15 min at 4 °C (EuroClone, Speed Master 14 R centrifuge, Italy). The supernatant was used for determination of the citrate synthase activity, and the protein concentration in the supernatant was determined spectrophotometrically by Bradford assay (Bio-Rad, USA), using a microplate reader (EnSpire, PerkinElmer, USA). Then, proteins were diluted in Tris-buffer 100 mM (pH 8.2) containing 5,5′-dithiobis-(2-nitrobenzoic) acid (DTNB, 100 µM) and acetyl-coenzyme A (100 µM). The assay was performed in 96-well plates (1 µg of proteins per well) and the reaction was initiated by addition of oxalacetate 500 µM. The absorption of the reaction product was measured spectrophotometrically at 30 °C and 412 nm every 30 s for 15 min. Citrate synthase activity was determined by comparing the activity in the samples to that of known concentrations of the isolated enzyme (Sigma-Aldrich, St. Louis, MI, USA). Citrate synthase activity was expressed in mU/µg protein. Data were analysed by a computer fitting procedure (software: GraphPad Prism 5.0).
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4

RNA Extraction from Yolk-Sac Fry

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Whole yolk-sac fry tissues were individually homogenized in ice using an Ultra Turrax homogeniser (T25 basic IKA-WERKE) at the speed setting of 4 for 10 s until they were homogeneous. Total RNA, DNA, and protein were extracted from whole yolk-sac fry (n = 16 per condition) using the QIAGEN AllPrep DNA/RNA/Prot Preparation Kit according to the manufacturer’s recommendations (Qiagen). The concentration of extracted RNA was analyzed using a spectrophotometer (Nanodrop ND1000, LabTech) by measuring absorbance at 260 nm. The quality of RNAs was checked with a Bioanalyzer (Agilent Technologies, Kista, Sweden), and 12 samples for each condition were selected for further analyses according to the RIN (RNA integrity number).
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5

Crude Membrane Preparation from T98G Cells

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For crude membrane preparation, T98G cells were grown to confluence and harvested using phosphate--buffered saline, supplemented with 0.04 % EDTA (w/v), pH 7.4. Cells were spun at 1000 g for 10 min and the pellet was suspended in 3 ml of ice--cold 5 mM Tris--HCl (pH 7.4) containing a protease inhibitor cocktail (1:100; Sigma--Aldrich). Cells were homogenised with a hand held Ultra--Turrax homogeniser (IKA Werke, Staufen, Germany). The homogenate was centrifuged at 48,000 g for 15 min at 4°C and the supernatant was discarded. The obtained pellet was resuspended in 10 ml of 50 mM Tris--HCl (pH 7.4; binding assay buffer), containing the protease inhibitor cocktail. The homogenate was re--pelleted by centrifugation at 48,000 g for 15 min at 4 °C. The pellet was washed once with binding assay buffer and an additional centrifugation step followed where the homogenate was spun at 48000 g for 15 min at 4 °C. Total protein in the resulting cell membrane pellet was determined using the bicinchoninic acid protein assay (Pierce Biotechnology Inc., Rockford, IL) according to the manufacturer's protocol.
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