The level of ethanol was measured as previously described [23 (link)] using an enzymatic detection kit UV-test for ethanol (RBiopharm, Darmstadt, Germany). A total of 1 x 106 cells were grown in the presence and absence of the argentilactone. Afterwards, the protein extract was obtained after cell lysis using glass beads and a bead beater apparatus (BioSpec, Oklahoma, USA) in 5 cycles of 30 sec. The cell lysate was subjected to centrifugation for 15 min, at 4°C, at 10,000 × g. The enzyme assay was performed in triplicate using the supernatant according to the manufacturer's instructions.
Bead beater apparatus
Manufactured by Biospec
Sourced in United States
The Bead Beater apparatus is a laboratory equipment used for the disruption and homogenization of biological samples. It utilizes high-speed agitation of samples containing beads to efficiently break down cells and tissues, enabling the extraction of intracellular components such as nucleic acids, proteins, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Uv test for ethanol, by R-Biopharm (3 mentions)
Bradford reagent, by Merck Group (2 mentions)
Glass bead, by Biospec (2 mentions)
Phenylhydrazine hcl, by Merck Group (1 mentions)
Glucose assay kit, by Abcam (1 mentions)
Pyruvate assay kit, by Abcam (1 mentions)
Atp colorimetric fluorometric assay kit, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
0.5 mm zirconia silica beads, by Biospec (1 mentions)
Rnase free dnase set 50, by Qiagen (1 mentions)
Protease inhibitor mix, by GE Healthcare (1 mentions)
Ab65622, by Abcam (1 mentions)
Dye binding protein assay kit, by Bio-Rad (1 mentions)
12 protocols using bead beater apparatus
1
Quantification of Cellular Ethanol Levels
2
Isocitrate Lyase Activity in Paracoccidioides
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Following Paracoccidioides Pb01, Pb03, Pb339, and PbEPM83 growth in the presence of acetate and glucose (both 100 mM), the cells were centrifuged at 1,500 × g, resuspended in a solution containing 20 mM Tris-HCl, pH 8.8, and 2 mM CaCl2 (Fonseca et al., 2001 (link)) and disrupted using glass beads and bead beater apparatus (BioSpec, Bartlesville, OK, USA) for 5 cycles of 30 s each while on ice. The cell lysate was centrifuged at 10,000 × g for 15 min at 4°C and the supernatant was quantified using Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA) (Bradford, 1976 (link)). The amount of protein used was 50 μg (Lima et al., 2014 (link)).
ICL activity was determined by measuring the formation of glyoxylate as its phenylhydrazone derivative (Ebel et al., 2006 (link)). Glyoxylate-phenylhydrazone formation was determined by measuring the absorbance at 324 nm with an extinction coefficient of 16.8 mM−1 cm−1 in a reaction mixture containing 2 mM threo-D,L-isocitrate (Sigma Aldrich), 2 mM MgCl2, 10 mM phenylhydrazine HCl (Sigma Aldrich), 2 mM dithiothreitol, and 50 mM potassium phosphate at pH 7.0. Specific activity was determined as the amount of enzyme required to form 1 μmol of glyoxylate- phenylhydrazone per min per mg of total protein. Statistical comparisons were performed using the Student's t-test and p-values ≤ 0.05 were considered statistically significant.
ICL activity was determined by measuring the formation of glyoxylate as its phenylhydrazone derivative (Ebel et al., 2006 (link)). Glyoxylate-phenylhydrazone formation was determined by measuring the absorbance at 324 nm with an extinction coefficient of 16.8 mM−1 cm−1 in a reaction mixture containing 2 mM threo-D,L-isocitrate (Sigma Aldrich), 2 mM MgCl2, 10 mM phenylhydrazine HCl (Sigma Aldrich), 2 mM dithiothreitol, and 50 mM potassium phosphate at pH 7.0. Specific activity was determined as the amount of enzyme required to form 1 μmol of glyoxylate- phenylhydrazone per min per mg of total protein. Statistical comparisons were performed using the Student's t-test and p-values ≤ 0.05 were considered statistically significant.
3
Quantification of Glucose and Pyruvate
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Glucose and pyruvate were measured using the Glucose Assay Kit (ab65333; Abcam) and Pyruvate Assay Kit (ab65342; Abcam) according to the manufacturer’s instructions (Liang et al., 2018 (link); Schnack et al., 2019 (link)). S. aureus strains were diluted 1:200 into TSB and incubated at 37°C. After 12 h, culture supernatant and cells were harvested. The cells were washed twice with phosphate-buffered saline (PBS) and adjusted to OD600 ≈ 1 with assay buffer and then were lysed with 0.1-mm glass-silica beads in a BeadBeater apparatus (BioSpec), followed by centrifugation to obtain the supernatant. The fluorescence signal of the supernatant was determined using a microplate reader with excitation at 535 nm and emission at 587 nm (Thermo VARIOSKAN LUX).
4
Quantification of Ethanol in Paracoccidioides Cells
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The concentration of ethanol was quantified by enzymatic detection kit according to the manufacturer's instruction (UV-test for ethanol, RBiopharm, Darmstadt, Germany). Ethanol is oxidized to acetaldehyde by the enzyme alcohol dehydrogenase, in the presence of nicotinamide-adenine dinucleotide (NAD). Acetaldehyde is quantitatively oxidized to acetic acid in the presence of aldehyde dehydrogenase, releasing NADH, which is determined by means of its absorbance at 340 nm. Paracoccidioides Pb01 yeast cells were subjected or not to carbon starvation, and 106 cells were used to assay. Briefly, cells were counted, centrifuged, and lysed using glass beads and bead beater apparatus (BioSpec, Oklahoma, USA) in 5 cycles of 30 sec, keeping the samples on ice. The cell lysate was centrifuged at 10,000× g for 15 min at 4°C and the supernatant was used for enzymatic assay according to the manufacturer's instructions. The concentrations of ethanol were obtained in triplicate.
5
Enzymatic Activity of L-Ornithine-N5-Oxygenase
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The enzymatic activity of L-ornithine-N5-oxygenase (SidA) was evaluated as previously described by Zhou et al. (1998 (link)) and Haas et al. (1999 (link)) with few modifications. Briefly, P. brasiliensis yeast cells were collected and suspended in 0.5 mM potassium phosphate buffer (pH 8.0). The suspension was transferred to tubes containing glass beads (425–600 μm) and submitted to vigorous mixing in a bead beater apparatus (BioSpec) for 5 cycles with intervals of 30 s on ice. The samples were centrifuged 10,000 x g for 15 min and the protein concentration in supernatants was determined with the (Bradford) reagent. To measure the enzymatic activity of SidA, equal amounts of proteins (50 μg) of both conditions, yeast cells incubated with (BPS) or (FOB), were used. The reaction mixture containing 40 μl of 0.5 mM potassium phosphate pH 8.0, 10 μl of 10 mM NADPH, 2 μl of 0.5 mM FAD, 50 μg of cell extract, 30 μl of 10 mM L-ornithine was incubated at 30 °C for 2 h and added of 100 μl of 0.2 M perchloric acid to stop the reaction. For control, the same amount of perchloric acid was added to one sample before incubation. The samples were centrifuged and the supernatants were used to determine the absorbance at 340 nm.
6
Intracellular ATP Levels in S. epidermidis Mutants
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The intracellular ATP levels in the ΔphoU1 and ΔphoU2 mutants were detected using an ATP colorimetric/fluorometric assay kit (catalog no. MAK190) from Sigma with modification for S. epidermidis . At the time points indicated above and in the relevant figures (when the OD600 was 0.5), the cells were pelleted by centrifugation at 4,000 × g at 4°C, washed with cold sterile PBS, resuspended in 1 ml of ATP extraction buffer within 10 min of initial pelleting of the culture, and lysed with 0.1 mm glass-silica beads in a BeadBeater apparatus (BioSpec). The resulting supernatant, obtained by centrifuging the samples at 20,000 × g for 15 min at 4°C, was filtered through a 10-kDa-cutoff filter, as suggested by the assay manufacturer, to remove enzymes that could otherwise deplete ATP.
7
RNA Extraction from Mouse Kidneys
8
Cytoplasmic Protein Extraction Protocol
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To obtain the cytoplasmic protein extract, the cells were centrifuged at 10,000 × g for 5 min, followed by washing with 1X PBS twice and the addition of Tris-Ca buffer (20 mM Tris–HCl pH 8.8; 2 mM CaCl2). This suspension was distributed in tubes containing an equal volume of glass beads (425–600 μm) to the volume of the cell pellet. The cells were disrupted by vigorous mixing in a bead beater apparatus (BioSpec) for 5 cycles for 30 s on ice. The cell lysate was centrifuged at 10,000 × g for 15 min at 4°C until no pellet formed. Protein content in the supernatant was quantified using Bradford reagent (Bradford, 1976 (link)) using bovine serum albumin as a standard.
9
Quantifying Ethanol Production in Paracoccidioides
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Ethanol concentration was determined using an enzymatic detection kit according to the manufacturer's instructions (UV-test for ethanol, RBiopharm, Darmstadt, Germany). Briefly, ethanol was oxidized to acetaldehyde by the enzyme alcohol dehydrogenase in the presence of nicotinamide-adenine dinucleotide (NAD). Acetaldehyde was quantitatively oxidized to acetic acid in the presence of aldehyde dehydrogenase, releasing NADH, which was determined by measuring the absorbance at 340 nm. Paracoccidioides Pb01, Pb03, Pb339, and PbEPM83 yeast cells were cultivated in minimal media containing acetate (100 mM) or glucose (100 mM), and 106 cells were assayed. Briefly, the cells were centrifuged and lysed using glass beads and a bead beater apparatus (BioSpec) in 5 cycles of 30 s while keeping the samples on ice. The cell lysate was centrifuged at 10,000 × g for15 min at 4°C and the supernatant was used for enzymatic assays according to the manufacturer's instructions. The concentrations of ethanol were determined in triplicate. Statistical analysis was performed using Student's t-test, and p ≤ 0.05 was considered significant.
10
Protein Extraction from Fungal Cells
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Mycelium cells were collected after 14 days of growth, mycelium-to-yeast transition cells after 3 days post temperature shift and yeast cells after 3 days of growth. Each sample phase was submitted to total protein extraction. Cells were collected by centrifugation at 10,000 × g for 10 min at 4°C and washed in sterile phosphate buffered saline (PBS; 1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl, and 2.5 mM KCl at pH 7.2). The cells were then resuspended in a buffer with 20 mM Tris–HCl at pH 8.8 and 2 mM CaCl2 and added a protease inhibitor mix (GE Healthcare, Uppsala County, Uppsala, Sweden) in order to prevent protein degradation. The samples were conditioned in proper tubes with glass beads and applied to a bead beater apparatus (BioSpec, Bartlesville, OK, United States) for 5 cycles of 30 s each, in order to disrupt cells and expose proteins to the solution. Samples were kept on ice to avoid protein denaturation. Then, the samples were centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was collected and the protein concentration was determined using Bradford reagent (Sigma-Aldrich, St. Louis, MO, United States). Bovine serum albumin (BSA) was used as standard.
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