To assess liver GS activity, samples were homogenised in a solution containing 50 mM imidazole-HCl, pH 6.8, 0.5 mM EDTA, 1 mM DTT, supplemented with protease inhibitors (Complete, Mini, EDTA-free, Roche, Basel, Swizterland) for 30 s on ice. Samples were then sonicated on ice (Sonicator Ultrasonic Processor XL, Misonix, Farmingdale, NY, USA) and centrifuged for 30 min at 12000 g at 4°C. After quantification (Bio-Rad Protein Assay), protein concentration of the supernatant was adjusted to 1 mg ml−1. An aliquot of 150 μl was used for the assay in a solution of 50 mM imidazole-HCl (pH 6.8), 50 mM Gln, 25 mM hydroxylamine, 25 mM sodium arsenate, 2 mM MnCl2, and 0.16 mM ADP. After incubation at 37 °C for 30 min, the reaction was stopped by the addition of a solution containing 2.42% FeCl3 and 1.45% TCA in 1.82% HCl. Precipitates were removed by centrifugation (2000 r.p.m. for 5 min) and supernatants were read at 540 nm using a spectrophotometer (Helios-γ Spectronic, Thermo Electron Corporation, Cambridge, UK). Values of GS activity were expressed as pmol of γ-glutamylhydroxamate min−1 μg−1 protein.
Sonicator ultrasonic processor xl
Manufactured by Bioventus
Sourced in United States
The Sonicator Ultrasonic Processor XL is a lab equipment designed for various applications that require high-intensity ultrasonic energy. It generates and delivers ultrasonic waves to process, mix, or disperse samples. The core function of this device is to convert electrical energy into high-frequency mechanical vibrations.
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7 protocols using sonicator ultrasonic processor xl
1
Quantitative Assay of Liver Glutamine Synthetase
2
Cellular Fractionation and Protein Analysis
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The cell fractions were prepared, as previously described [63 (link)], and then adapted to cells in a six-well format. Briefly, the cells were washed with PBS, harvested by scraping, and transferred to a 1.5 mL tube. Cells were pelleted by centrifugation 30 s on a bench-top centrifuge. The pellet was resuspended in 108 µL ice-cold 0.1% NP-40 in PBS and triturated five times using a 200 µL micropipette, and then re-centrifuged for 30 s. For the cytosolic fraction, 36 µL of the supernatant was collected and mixed with 12 µL of 4× Laemmli sample buffer. For the nuclear fraction, the remaining material was re-centrifuged for 30 s and the pellet was washed once with 0.1% NP-40 in PBS, resuspended in 110 µL 1× Laemmli sample buffer, and sonicated twice for 5 s at level 2 using a Sonicator ultrasonic processor XL (Misonix, Inc., Farmingdale, NY, USA). After the addition of β-mercaptoethanol, the samples were heated at 95 °C for 5 min, loaded onto an AnyKD precast MiniProtean TGX Stain Free Gel, and processed, as described above.
3
Optimization of Enzyme Purification from HepG2
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HepG2 cells were grown to 90% confluence (1.9 × 106 cells) in D-MEM culture medium, containing 10% (v/v) heat-inactivated fetal calf serum, in the presence of 1% (v/v) penicillin-streptomycin on a dish coated with collagen (Cellmatrix I) at 37 °C with 5.0% CO2. The medium was replaced every day. Cells were collected by scraping with 0.1% EDTA/PBS then 15 mM Tris/HCl buffer (pH 7.0, 3 mL) and glass beads (diameter <106 μm, 50 mg) were added. The cell suspension was sonicated for 3 min at 3 intervals on ice using a Sonicator Ultrasonic Processor XL (Misonix, New York, NY, USA) followed by ultracentrifugation (11,000g) for 15 min at 4 °C. The supernatant was collected and dialyzed in 70% ammonium sulfate overnight at 4 °C. Then, the precipitate was collected by removing the supernatant after centrifugation (11,000g) for 20 min at 4 °C. The residue was dissolved in 2 mL of 50 mM citrate buffer (pH 5.0, 6.0, 6.9) and dialyzed in 50 mM citrate buffer (pH 5.0, 6.0, 6.9) for 6 h at 4 °C. Finally, the obtained solution was brought to an amount of 2 mL by adding 50 mM citrate buffer (pH 5.0, 6.0, 6.9).
4
Quantifying Biofilm Viability Dynamics
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Following the initial 24 hour incubation period, the biofilm-coated peg lid was removed from the cassette and immersed in a 96-well microliter plate containing test treatments. Treatments were performed twice daily, based on usage instructions of the product. Five treatments were performed over the course of 60 hours. Biofilm-coated peg lids were reimmersed into a fresh saliva media mixture following each treatment course. Immediately after the final treatment, biofilms were rinsed and harvested via sonication (Sonicator Ultrasonic Processor XL, MISONIX Inc.). Aliquots were washed with 0.1% peptone water and analyzed for ATP (Berthold luminometer (Bad Wildbad, Germany) and Celsis rapid screen ATP bioluminescence reagents (Catalog number 1230941)). Results were reported as log values of relative light units (RLU) per well. The RLU value represented the amount of viable cells.
5
Preparation of Soluble and Total Tachyzoite Extracts
6
Arsenic Stress Response in Thermus thermophilus
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T. thermophilus HB27 cultures were grown aerobically at 70°C in TM medium, as previously described (24 (link)). Once the cultures reached an optical density at 600 nm (OD600) of 0.5, they were treated either with 8 mM NaAsO2 or with 12 mM NaH2AsO4 (Sigma) (the used concentrations were below the previously reported MIC values for arsenate and arsenite) (22 (link), 23 (link)) or they remained untreated. Samples were harvested from each culture, either immediately after treatment or 60 min posttreatment. The samples were centrifuged, the precipitates were resuspended in phosphate buffer (20 mM Na3PO4, pH 7.5) supplemented with protease inhibitor cocktail (Thermo Scientific), and the resuspended cells were lysed by sonication (10 cycles of 30 s on/30 s off, 40% power; Misonix Sonicator Ultrasonic Processor XL). The lysates were centrifuged and the cell extracts (CFE) used for pulldown assays.
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7
Purification and Quantification of Neutrophil Arginase
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Purified peripheral blood human PMNs were resuspended in PBS (40 × 106 cells/ml), sonicated for 3 min (amplitude 80) in a Sonicator Ultrasonic Processor XL (Misonix, Inc. New Highway, Farmingdale, NY), and centrifuged at 20,000g for 30 min at 4 °C, as previously described9 (link). Then, the supernatant was filtered (0.2 µm), protein concentration and arginase activity were determined, and aliquots were frozen at − 80 °C until use as previously described9 (link).
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