Cryptotanshinone was acquired from Sigma (C5624–5MG). 5 nM Mb13 monobody was incubated with 20 µg/ml streptavidin-coupled Dynabeads M280 (Invitrogen) for 30 min on ice in BSS buffer (50 mM Tris–HCl, 150 mM NaCl, pH 8, 1 mg/ml BSA, 1 mM DTT, and 1 mM EDTA). The beads were then washed and blocked with 1 mM biotin for 30 min, followed by another wash and resuspension in BSS buffer. 200 nM SHP2 PTP was pre-incubated with or without 100 µM cryptotanshinone for 30 min at 30 °C in BSS buffer. Ten microliters of the Mb13-bead solution were then transferred to a well of a 96-well filter plate (MultiScreenHTS HV, 0.45 µm pore size; Millipore) and drained by vacuum. Twenty µl of the SHP2 PTP mixture were then transferred to the wells of the filter plate containing the drained beads and the plate was incubated at 30 °C with shaking for 30 min. The wells were drained and washed twice with ice-cold BSST (BSS buffer containing 0.1% Tween 20). After draining, 20 µl of 10 µg/ml of Dylight 650-conjugated to streptavidin (Thermo) in BSS was added to each of the wells. After incubation on ice with shaking for 30 min, the beads were washed with BSST twice in the same manner described as above. The beads were resuspended in 200 µl BSST and the fluorescence emission in the Red2 channel was analyzed for 5000 events on a Guava EasyCyte 6/L flow cytometer (Millipore).
Multiscreenhts hv
Manufactured by Merck Group
Sourced in United States
The MultiScreenHTS HV is a high-volume, automated 96-well filtration system designed for rapid sample processing in high-throughput screening applications. It provides efficient liquid handling and filtration for a wide range of assay formats, supporting research and development workflows in the life sciences industry.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads m 280, by Thermo Fisher Scientific (2 mentions)
Dylight 650 conjugated to streptavidin, by Thermo Fisher Scientific (2 mentions)
Guava easycyte 6 l flow cytometer, by Merck Group (2 mentions)
Dextran coated charcoal, by Merck Group (1 mentions)
Ls6500, by Beckman Coulter (1 mentions)
Winnolin version 6, by Pharsight (1 mentions)
Precellys 24, by Bertin Technologies (1 mentions)
96 deep well plate, by Avantor (1 mentions)
6 protocols using multiscreenhts hv
1
Cryptotanshinone Inhibition of SHP2 PTP
2
Saturation Binding Assay for ERRγ-LBD
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A saturation binding assay was conducted [27] (link) using [3H]BPA. The reaction mixture was incubated at 4°C for 2 h with the receptor proteins—GST-fused wild-type ERRγ-LBD or its mutants—in 100 µl of binding buffer [10 mM HEPES (pH 7.5), 50 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 2 mM CHAPS, and 2 mg/ml γ-globulin]. The assay was performed with or without the addition of unlabelled BPA (final concentration of 1.0×10-5 M) to quantify the specific and nonspecific binding. After incubation with 100 µl of 1% dextran-coated charcoal (DCC) (Sigma-Aldrich) [28] (link) in PBS (pH 7.4) for 10 min at 4°C, the DCC-absorbed free radio-ligand was removed by the direct vacuum filtration method using a 96-well filtration plate (MultiScreenHTS HV, 0.45 µm pore size; Millipore, Billerica, MA, USA) for the bound/free separation [27] (link). Radioactivity was determined on a liquid scintillation counter (LS6500; Beckman Coulter, Fullerton, CA, USA).
The data on the specific binding of [3H]BPA were first assessed by means of Scatchard plot analysis [29] (link). Then, these data were applied to a one-site binding hyperbola nonlinear regression analysis by the software package Prism (GraphPad Software Inc., La Jolla, CA, USA) to measure changes in the receptor density Bmax and equilibrium dissociation constant Kd. The saturation binding assay was performed at least three times.
The data on the specific binding of [3H]BPA were first assessed by means of Scatchard plot analysis [29] (link). Then, these data were applied to a one-site binding hyperbola nonlinear regression analysis by the software package Prism (GraphPad Software Inc., La Jolla, CA, USA) to measure changes in the receptor density Bmax and equilibrium dissociation constant Kd. The saturation binding assay was performed at least three times.
3
Cryptotanshinone Inhibition of SHP2 PTP
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Cryptotanshinone was acquired from Sigma (C5624–5MG). 5 nM Mb13 monobody was incubated with 20 µg/ml streptavidin-coupled Dynabeads M280 (Invitrogen) for 30 min on ice in BSS buffer (50 mM Tris–HCl, 150 mM NaCl, pH 8, 1 mg/ml BSA, 1 mM DTT, and 1 mM EDTA). The beads were then washed and blocked with 1 mM biotin for 30 min, followed by another wash and resuspension in BSS buffer. 200 nM SHP2 PTP was pre-incubated with or without 100 µM cryptotanshinone for 30 min at 30 °C in BSS buffer. Ten microliters of the Mb13-bead solution were then transferred to a well of a 96-well filter plate (MultiScreenHTS HV, 0.45 µm pore size; Millipore) and drained by vacuum. Twenty µl of the SHP2 PTP mixture were then transferred to the wells of the filter plate containing the drained beads and the plate was incubated at 30 °C with shaking for 30 min. The wells were drained and washed twice with ice-cold BSST (BSS buffer containing 0.1% Tween 20). After draining, 20 µl of 10 µg/ml of Dylight 650-conjugated to streptavidin (Thermo) in BSS was added to each of the wells. After incubation on ice with shaking for 30 min, the beads were washed with BSST twice in the same manner described as above. The beads were resuspended in 200 µl BSST and the fluorescence emission in the Red2 channel was analyzed for 5000 events on a Guava EasyCyte 6/L flow cytometer (Millipore).
4
Human DP1 Receptor Binding Assay
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Membranes were prepared from 1321 NI astrocytoma cells (Sigma‐Aldrich, Gillingham, Dorset, UK)) stably expressing human recombinant DP1 receptors (Woodward et al., 2007 , Wang et al., 2016 ). For assays, membranes (protein 15 μg per well) expressing the human DP1 receptor were incubated in Millipore MultiscreenHTS‐HV (0.45 μm) plates containing assay buffer (50 mM Tris, 5 mM MgCl2, 10 μg·mL−1 saponin and 10 mM indomethacin), [3H] PGD2 3.2 nM and the test compounds (0.1 to 10 000 nM) at pH 7.4. Incubations were performed at 25°C for 60 min to allow equilibrium to be reached. Non‐specific binding was determined in the presence of 10 μM PGD2. Bound and free radiolabel were separated by vacuum manifold filtration using an appropriate wash buffer, and bound radiolabel was determined by scintillation counting. The affinity or pKi of each compound was calculated from the concentration causing 50% radioligand displacement (IC50) using the Cheng–Prusoff equation as previously described.
5
Pharmacokinetic Assessment of SDM-878
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SDM‐878 was administered intravenously at a dose of 1 mg/kg to the fed rats or orally at a dose of 30 mg/kg to the rats fasted overnight. Blood samples were collected at appropriate time intervals after the administration of SDM‐878 and then centrifuged at 2150 g for 15 minutes at 4°C to obtain plasma. Each plasma sample was centrifuged at 540 g for 5 minutes at 4°C to yield supernatants before being filtrated using MultiScreen HTS HV (Millipore Corp) and analyzed by liquid chromatography with tandem mass spectrometry (LC‐MS/MS). To assess brain penetration, SDM‐878 was administered to the fed rats orally at doses of 30 and 100 mg/kg. Blood samples were collected 30 minutes after the administration, after which the rats were immediately euthanized, and their brains were collected. The collected brain samples were homogenized in 5 times the brain volume of H2O using a Precellys24 (Bertin Technologies SAS). An internal standard was added, and the homogenates were centrifuged at 3000 rpm for 5 minutes at 4°C to yield supernatants for analysis by LC‐MS/MS. Pharmacokinetic parameters were calculated via noncompartmental analysis using WinNolin, version 6.2 (Pharsight).
6
High-Throughput Screening of Mutant Taq Polymerases
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Colonies expressing mutant Taq polymerases were randomly picked, replicated, and then grown overnight at 30°C in 96-deep well plates (VWR) containing 750 μl LB/CAM. Overnight cultures (30 μl) were inoculated into fresh media, induced with anhydrotetracycline at OD600nm of 0.3–0.5, and grown overnight with shaking at 30°C. Cells were collected and used to prepare lysates for direct PCR screening or for affinity protein purification (see below). Cell pellets were re-suspended in 50 μl Tris pH 8 containing 4 mg/ml lysozyme, and incubated at 37°C for 10 min to disrupt cell walls and at 75°C for 15 min to inactivate E. coli protein. Lysates were clarified by centrifugation for 15–30 min at 4000 RPM through a 96-well filter plate (Millipore, Multiscreen HTS, HV).
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