VICs were isolated from AVs removed for severe stenosis using tissue lysis with 2% collagenase (Sigma-Aldrich, St Louis, MO, USA). Protein extraction and proteolysis were performed with the methanol-chloroform method and trypsin [(Gold Grade; Promega, Wisconsin)/RapiGest procedure (Waters, Milford, MA, USA)], respectively, as previously published (18 (link)). Fifteen micrograms of protein was used per sample. The tryptic peptides were desalted using Oasis Hlb 1cc (10 mg) columns (Waters, Milford, MA, USA), and dried with a tabletop speed vacuum (SPD1010, Thermo Fisher Scientific, USA). After resuspension in 40 μl of 5% mass spectrometry grade acetonitrile (Thermo Fisher Scientific, USA) and 5% formic acid (Sigma-Aldrich, St Louis, MO, USA), the tryptic peptide samples were analyzed by liquid chromatography–mass spectrometry.
Spd1010
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SPD1010 is a high-performance vacuum concentrator designed for the efficient removal of solvents and other volatile compounds from liquid samples. It features a compact design, digital temperature control, and a liquid nitrogen cold trap to ensure reliable sample protection and recovery.
Automatically generated - may contain errors
Lab products found in correlation
Allegra x 14r, by Beckman Coulter (2 mentions)
Mass spectrometry grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Rapigest, by Waters Corporation (1 mentions)
Formic acid, by Merck Group (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Bagmixer 400, by Interscience (1 mentions)
4 protocols using spd1010
1
Isolation and Proteomic Analysis of Valvular Interstitial Cells
2
Extraction and Drying of Rice Bran Antioxidants
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RBE was prepared as described previously (Kumar et al. 2012 ). Briefly, 4 g of finely ground, heat‐stabilized Calrose rice bran (USDA‐ARS Rice Research Unit, Stuttgart, AK) was extracted in 42·6 ml of 80% methanol. The mixture was vortexed (232 Vortexer Fisher Scientific, Pittsburgh, PA, USA) on a high power setting for 5 min, incubated at −80°C overnight and centrifuged (Beckman Coulter Allegra X‐14R, Indianapolis, IN, USA) at 3724 g for 5 min. The supernatant was collected, and kept at −80°C until it could be dried in a speedvac concentrator (SPD1010; Thermo Scientific, Pittsburgh, PA, USA) at 45°C, with the heating time for 5 min, and a vacuum pressure of 7·5 torrs.
3
Peptide Extraction from Samples
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Peptide extraction was conducted using a modified version of the method by Gallego et al. (2016) . Ten grams of sample were homogenized with 50 mL of 0.01 N HCl for 3 min in a stomacher (BagMixer 400, Interscience, Saint Nom, France). The homogenate was centrifuged at 10,000 × g for 30 min at 4°C and filtered through glass wool. Three volumes of ethanol were mixed with the filtrate, and it was stored for 24 h at 4°C. The mixture was centrifuged at 10,000 × g for 30 min at 4°C, and the supernatant was lyophilized using a vacuum evaporator (SPD1010, Thermo Fisher Scientific Inc). Lyophilized samples were dissolved in 5 mL of 0.01 N HCl, neutralized to pH 7.0 using NaOH, and filtered using a 0.45-µm nylon membrane filter (Millipore Corp., Bedford, MA). The filtrates were centrifuged in centrifugal filter-containing tubes (Amicon Ultra-15 Centrifugal Filter Unit, Millipore) at 10,000 × g for 30 min.
4
Rice Bran Extract Preparation and Supplementation
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Rice bran extract was prepared using heat-stabilized Calrose rice bran (USDA-ARS Rice Research Unit, Stuttgart, AK), as previously described (Forster et al., 2013 (link)). Heat stabilization of rice bran was completed in a commercial dryer for 30 min at 110°C. In total, 4 g of rice bran was extracted in 42.6 mL of 80% aqueous solution of ice-cold (−80°C) methanol, vigorously vortexed for 5 min (232 Vortexer Fisher Scientific, Pittsburgh, PA, USA), incubated overnight at −80°C, and centrifuged at 4,000 g for 5 min (Beckman Coulter Allegra X-14R). The supernatant was collected and dried in a speedvac concentrator (SPD1010, Thermo Scientific, Pittsburgh, PA) at 45°C for approximately 48 h. To prepare MRS + rice bran extract, 100 μg of rice bran extract was added to 1 mL of MRS broth and autoclaved using a sterilization time of 45 min. Broth was stored at 4°C until use. The concentration of MRS + rice bran extract was the same as previous dose response studies of MRS + rice bran extract broth on S. Typhimurium growth (Nealon et al., 2017a (link)).
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