A 14C collagen solution, prepared as described above, was used for the gelatinase assay after heating at 45 °C for 15 min to form 14C-gelatin [44 (link),45 (link),46 (link)]. A quantity of 50 μL of the substrate (2.69 mg/mL gelatin) was added to 50 μL buffer (0.1 M Tris-HCl, pH 7.6) and 100 μL of the sample. The incubation mixture also contained 0.15 M NaCl and 5 mM CaCl₂. After incubation for 120 min at 37 °C, 100 μL of 50% (w/v) trichloroacetic acid was added, and the tube was placed on ice for 5 min and then centrifuged for 4 min in a Beckman microfuge. A quantity of 200 μL of the supernatant was counted for radioactivity in 9 mL of scintillation fluid. For the blanks, EDTA (10 mM) was included in the incubation mixture. Enzyme activity was expressed as dpm per mg protein per hour.
Microfuge
Manufactured by Beckman Coulter
Sourced in United States
The Microfuge is a laboratory centrifuge designed for the separation of small sample volumes. It is used to separate and concentrate materials, such as proteins, nucleic acids, or cells, based on their density differences. The Microfuge's core function is to provide a controlled centrifugal force to enable efficient separation and purification of samples in a variety of laboratory applications.
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Lab products found in correlation
Allegra centrifuge, by Beckman Coulter (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Tl 100 ultracentrifuge, by Beckman Coulter (1 mentions)
Coomassie brilliant blue r 250, by National Diagnostics (1 mentions)
V700 scanner, by Epson (1 mentions)
Tla100 rotor, by Beckman Coulter (1 mentions)
Bradford assay, by Avantor (1 mentions)
10 protocols using microfuge
1
Gelatinase Activity Quantification Protocol
2
Cell Lysis and Clarification Protocol
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MT-2 cells were collected and washed three times by pelleting at 200 × g for 5 min at 4°C in an Allegra centrifuge (Beckman) and resuspending cells in ice-cold PBS. Cell pellets were lysed with 250 μl lysis buffer (20 mM HEPES [pH 7.9], 14 mM potassium acetate, 1 mM MgCl2, 0.3% NP-40), followed by shearing 20× with a 20-gauge needle. Cell lysate was then clarified 200 × g for 10 min at 4°C in an Allegra centrifuge (Beckman). The supernatant was transferred to a fresh tube and further clarified by centrifugation at 18,000 × g for 10 min at 4°C in a Microfuge (Beckman), and the supernatant was transferred to a fresh tube.
3
Cell Line Lysis and Extraction
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U2OS, HeLa, and HEK293 cell lines were purchased from ATCC. Cells were trypsinized, washed, and lysed with buffer containing 50 mM Tris, 0.5% NP-40, 150 mM NaCl, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 5 µg/ml leupeptin. After incubation for 30 min on ice, the extracts were centrifuged at 14,000 rpm in a microfuge (Beckman Coulter) for 20 min at 4°C, and the supernatant was collected.
4
Quantification of Brain Neurotransmitters
5
Extracellular pro-PaAP processing in P. aeruginosa
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P. aeruginosa strains FRD2, FRD740 and FRD1185 were grown in tryptic soy broth without glucose for 24 h. Bacteria were removed by centrifugation and the clear supernatants were stored in aliquots at -20°C until use, usually up to 3 days. In an experiment designed to follow extracellular pro-PaAP processing, FRD2 bacteria were harvested at the late logarithmic phase of growth (OD at 660 nm of ~2) by centrifugation (6,000 g; 20 min, 4°C). Pelleted bacteria were washed with sterile medium, re-suspended in the original volume of fresh medium and incubated at 37°C for different time intervals. At each time point, a sample removed from the culture suspension was centrifuged immediately (Beckman microfuge; 1 min; room temperature) to remove bacterial cells. The supernatants thus obtained were re-centrifuged (4 min), supplemented with bovine serum albumin (100 μg; carrier) and proteins in the supernatants were precipitated with trichloroacetic acid (TCA). After washing, the pellets were solubilized and prepared for SDS-PAGE as described [41 (link)].
6
Standardized P. aeruginosa Culture Protocol
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Overnight cultures of all P. aeruginosa strains used in this study were standardized to OD600 0.5 and 1:50 diluted in 10 mL LB with or without antibiotics (sub-MIC 12.5). After 24 h incubation at 37 °C, the cell pellets and supernatant were collected and filter-sterilized. For cells extract cell P. aeruginosa cells pellet were harvested by centrifugation (Beckman microfuge; 10 min) at 4 °C, then cells were suspended in lysis buffer and kept at −20 °C for further analysis (Kessler et al., 1998 (link)). All experiments were performed three times as triplicates.
7
Enzymatic Modification of Peptides
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2-Hydroxyisobutyrylated, acetylated, or succinylated peptides (50 μM) with 1 μM CobB were incubated in a reaction buffer [50 mM tris-HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM DTT, and 1 mM NAD+ (pH 8.5)] for 2 hours at 37°C. The reaction was then quenched with 1 volume of 10% (v/v) trifluoroacetic acid and spun for 10 min at 18,000g (Beckman Coulter Microfuge) to separate the enzyme from the reaction. The sample was separated by a 50-min HPLC gradient [linear gradient from 2 to 35% HPLC buffer B (0.1% formic acid in acetonitrile) for 35 min and then to 90% buffer B for 15 min]. The supernatant was then analyzed using nano-HPLC–MS/MS. The HPLC elute was electrosprayed directly into an Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA).
8
Actin Bundling and Binding Assay
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F-actin (10 μM) was incubated with various concentrations of UNC-87A or UNC-87B in F-buffer (0.1 M KCl, 2 mM MgCl2, 1 mM dithiothreitol [DTT], 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid–KOH, pH 7.5) for 30 min at room temperature. The reactions were centrifuged either at low speed (18,000 rpm for 10 min using a Beckman Microfuge) to examine actin bundling or at high speed (80,000 rpm for 20 min using a Beckman TL-100 ultracentrifuge with a TLA-100 rotor) to examine F-actin binding. The supernatants and pellets were adjusted to the same volumes and analyzed by SDS–PAGE. Gels were stained with Coomassie Brilliant Blue R-250 (National Diagnostics, Atlanta, GA) and scanned by an Epson V700 scanner at 300 dots/inch, and the band intensity was quantified by ImageJ (National Institutes of Health, Bethesda, MD).
9
In Vitro TT-Loaded PCL Nanoparticle Stability
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Ten milligrams of TT-loaded PCL NPs (60 nm or 450 nm) was dissolved in 1 ml of either PBS (pH 7.2), PBS (pH 4.0), or double-distilled water (pH 6.0) and kept at 37°C for 7, 14, 21, 28, and 30 days. After the indicated time points, the Microfuge® (Beckman Coulter, Pasadena, CA, USA) tubes were centrifuged at 15,000 rpm, and the supernatants were assayed for protein content using a Bradford assay (Amresco, Solon, OH, USA). Values were obtained from the standard curve obtained with bovine serum albumin (BSA) as reference protein. Void PCL NPs were used as negative controls.
10
Analyzing Ce-myosin Binding Kinetics
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Ce-myosin (0.19 μM) was incubated with various concentrations of UNC-87A or UNC-87B in a buffer containing 27 mM KCl, 1 mM MgCl2, 1 mM DTT, and 20 mM imidazole-HCl, pH 7.5, for 30 min at room temperature. The reactions were centrifuged at 18,000 × g for 30 min using a Beckman Microfuge. The supernatants and pellets were fractionated, adjusted to the same volumes, and analyzed by SDS–PAGE. Quantitative analysis was done in the same manner as described for actin sedimentation assays.
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