PTP activity was assayed using p-nitrophenyl phosphate (pNPP) as a substrate in DMG buffer (50 mM DMG, pH 7.0, 1 mM EDTA, 150 mM NaCl, 2 mM DTT, 0.1 mg/mL BSA) at 25 °C. The assays were performed in 96-well plates. To determine the IC50 values, the reaction was initiated by the addition of enzyme (final concentration = 10 nM) to a reaction mixture (0.2 mL) containing pNPP (at a final concentration close to the Kms of tested enzymes, in specific: 0.05 mM for CDC-14A; 0.5 mM for FAP-1; 2 mM for TC-PTP, PTP1B, STEP, LWM-PTP, and PTPα; 3 mM for SHP1; 4 mM for Laforin; 5 mM for LYP, CD45, and VHR; 6 mM for PTP-MEG2 and HePTP) with various concentrations of inhibitors. The reaction rate was measured using a SpectraMax Plus 384 Microplate Spectrophotometer (Molecular Devices, San Jose, CA, USA). Data were fitted using the SigmaPlot Enzyme Kinetics Module (Systat Software, Inc., San Jose, CA, USA).
Spectra max plus 384 microplate spectrophotometer
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax Plus 384 Microplate Spectrophotometer is a versatile laboratory instrument designed for absorbance-based measurements. It features a 384-well microplate format and can perform a range of spectrophotometric analyses, including endpoint, kinetic, and spectral scanning. The instrument utilizes a xenon flash lamp as the light source and incorporates a monochromator for wavelength selection. The SpectraMax Plus 384 supports a wavelength range of 190 to 850 nanometers.
Automatically generated - may contain errors
Lab products found in correlation
Sigmaplot enzyme kinetics module, by Grafiti LLC (2 mentions)
Quantikine hs elisa human il10, by RnD Systems (2 mentions)
Human interleukin 35 il35 elisa kit, by Nordic Biosite (2 mentions)
Softmax pro 7, by Molecular Devices (2 mentions)
Human tgf beta 1 elisa kit, by Abcam (2 mentions)
Non treated 96 well plates, by Genesee Scientific (2 mentions)
Celltiter 96 aqueous one solution reagent, by Promega (1 mentions)
Celltiter 96, by Promega (1 mentions)
Celltiter glo 2.0 cell viability assay, by Promega (1 mentions)
A23187, by Merck Group (1 mentions)
M6a rna methylation quantification kit, by Abcam (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Celltiter 96 aqueous one solution, by Promega (1 mentions)
Du 640 uv vis spectrophotometer, by Beckman Coulter (1 mentions)
Nanodrop 2000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Thermomixer comfort, by Eppendorf (1 mentions)
Fluorolog 3 spectrofluorometer, by Horiba (1 mentions)
Spectramax gemini microplate spectrofluorometer, by Molecular Devices (1 mentions)
20 protocols using spectra max plus 384 microplate spectrophotometer
1
Phosphatase Inhibitor Assay Protocol
2
Anti-HeV-sG Antibody Quantification ELISA
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Testing for the presence of anti-HeV-sG antibodies was performed according to a characterized method (Profectus BioSciences SOP 339, “Quantification of Anti-HeV-sG Antibodies in Non-Human Primate Sera by ELISA”) in a nonregulated (non-GLP and non-GMP) discovery-based laboratory. A purified monoclonal anti-HeV-sG antibody, m102.4, that also binds to NiV-sG was included as a positive control with established acceptance criteria on each ELISA plate. In brief, each serum sample or control antibody was tittered onto 96-well plates coated with purified HeV-sG (or NiV-sG) protein. Anti-HeV-sG (or anti-NiV-sG) antibodies from the samples were captured on the plate and detected using a horseradish peroxidase (HRP)-conjugated goat anti-monkey IgG secondary antibody. TMB peroxidase substrate was added to produce a chromogenic reaction that was proportional to the amount of antibody on the plate. After stopping the reaction with acid, the 450 nm absorbance of each well was read using a SpectraMaxPlus 384 microplate spectrophotometer (Molecular Devices, LLC, Silicon Valley, California). Binding curves and half-maximum antibody binding titers were calculated using SoftMax Pro 5.4 microplate data analysis software (Molecular Devices).
3
Cell Viability Assay of Nucleic Acid Treatments
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Twenty-four
hours prior to treatment, cells were seeded (1 × 104/well) into a 96-well plate. Treatments (CSpp at various N/P ratios,
PEI/pDNA polyplexes, and pDNA alone) were added in a volume of 100
μL/well (equivalent to 1 μg of pDNA) of serum-free medium.
Without aspiration, 100 μL of fresh complete medium was added
after 4 h of incubation. After 48 h, the treatment was aspirated and
replaced with 100 μL of fresh complete medium and 20 μL
of the MTS tetrazolium compound or CellTiter 96 Aqueous One Solution reagent (Promega Corporation, Madison, WI). The plate
was incubated at 37 °C at 5% CO2 and incubated for
1–4 h, and then the absorbance was recorded at 490 nm using
a Spectra Max plus 384 Microplate spectrophotometer (Molecular Devices,
Sunnyvale, CA). Relative cell viability values are expressed as the
percentage of UV absorbance from wells containing treated cells compared
to the control wells containing live cells treated with phosphate
buffered saline (PBS).
hours prior to treatment, cells were seeded (1 × 104/well) into a 96-well plate. Treatments (CSpp at various N/P ratios,
PEI/pDNA polyplexes, and pDNA alone) were added in a volume of 100
μL/well (equivalent to 1 μg of pDNA) of serum-free medium.
Without aspiration, 100 μL of fresh complete medium was added
after 4 h of incubation. After 48 h, the treatment was aspirated and
replaced with 100 μL of fresh complete medium and 20 μL
of the MTS tetrazolium compound or CellTiter 96 Aqueous One Solution reagent (Promega Corporation, Madison, WI). The plate
was incubated at 37 °C at 5% CO2 and incubated for
1–4 h, and then the absorbance was recorded at 490 nm using
a Spectra Max plus 384 Microplate spectrophotometer (Molecular Devices,
Sunnyvale, CA). Relative cell viability values are expressed as the
percentage of UV absorbance from wells containing treated cells compared
to the control wells containing live cells treated with phosphate
buffered saline (PBS).
4
Biocompatibility Evaluation of IP-L-780
5
Phosphatase Inhibitor Assay Protocol
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PTP activity was assayed using p-nitrophenyl phosphate (pNPP) as a substrate in DMG buffer (50 mM DMG, pH 7.0, 1 mM EDTA, 150 mM NaCl, 2 mM DTT, 0.1 mg/mL BSA) at 25 °C. The assays were performed in 96-well plates. To determine the IC50 values, the reaction was initiated by the addition of enzyme (final concentration = 10 nM) to a reaction mixture (0.2 mL) containing pNPP (at a final concentration close to the Kms of tested enzymes, in specific: 0.05 mM for CDC-14A; 0.5 mM for FAP-1; 2 mM for TC-PTP, PTP1B, STEP, LWM-PTP, and PTPα; 3 mM for SHP1; 4 mM for Laforin; 5 mM for LYP, CD45, and VHR; 6 mM for PTP-MEG2 and HePTP) with various concentrations of inhibitors. The reaction rate was measured using a SpectraMax Plus 384 Microplate Spectrophotometer (Molecular Devices, San Jose, CA, USA). Data were fitted using the SigmaPlot Enzyme Kinetics Module (Systat Software, Inc., San Jose, CA, USA).
6
Treg-specific Cytokine Quantification
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The Quantikine HS ELISA Human IL10 (RnD Systems, cat. HS100C), Human TGF beta 1 ELISA kit (Abcam, cat. ab100647) and Human Interleukin 35 (IL35) ELISA Kit (Nordic BioSite, cat. EKX-6FHVKH-96) were used to measure the amount of Treg-specific cytokines in Treg suppression assay supernatant, according to instructions from the manufacturers. For IL35, samples were diluted 1:1 in sample dilution buffer, for IL10 and TGF- , samples were handled according to manufacturer’s protocols. Absorbance was read at A450 nm (IL35 and TGF- ), and A490 nm (IL10) using the SpectraMax plus 384 Microplate Spectrophotometer and SoftMax Pro 7.1 software (Molecular Devices).
7
REST-Knockdown Cell Viability Assay
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SU-DIPG-IV and /or SU-DIPG-XIII cells were plated in a 96-well plate at a density of 6,000 cells/well in 100μL complete media. At various times (24, 48 or 72h) after lentiviral transduction with control shRNA or REST-shRNA, 50μL of media was decanted, and 25ng of 98% Thiazolyl Blue Tetrazolium Bromide (MTT) (Cat# M2128-1G; Sigma, St. Louis, MO) was added. Cells were incubated for 2-4 hours before 100 μl of DMSO was added to each well and triturated to uniformly suspend the MTT precipitate. Plates were read for absorbance (570/650 nm) with a SPECTRAmax®PLUS384 Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA).
8
Ubiquinol Cytotoxicity in HCEC-B4G12 Cells
9
Sacculi Hydrolysis Assay Protocol
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Sacculi was prepared from stationary-phase cells of S. aureus strain HG003 using a previously published protocol52 (link), with the following modifications. Volumes of solutions were scaled appropriately for the number of cells harvested. After 1 M HCl treatment, the pellets were washed with dH2O, flash-frozen, and lyophilized to yield purified sacculi.
A 0.5 mg/mL solution of sacculi was prepared in reaction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% DDM, 60 μM Zn(OAc)2). The sacculi hydrolysis assay was set up in non-treated 96-well plates (Genesee Scientific). To each well, 150 μL of sacculi solution was added. Lysostaphin (dissolved in dH2O) or the purified LytH-ActH complex (stored in 50 mM HEPES, pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% DDM) was mixed with the sacculi substrate. Reactions were prepared in triplicate using 0–125 nM of purified protein per reaction. The plate was covered with a lid and incubated at 25°C with shaking. Absorbance (OD600) was recorded at 20-min intervals for 20 hr using a SpectraMax Plus 384 microplate spectrophotometer (Molecular Devices). Hydrolysis of sacculi was monitored over time as a decrease in absorbance.
A 0.5 mg/mL solution of sacculi was prepared in reaction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% DDM, 60 μM Zn(OAc)2). The sacculi hydrolysis assay was set up in non-treated 96-well plates (Genesee Scientific). To each well, 150 μL of sacculi solution was added. Lysostaphin (dissolved in dH2O) or the purified LytH-ActH complex (stored in 50 mM HEPES, pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% DDM) was mixed with the sacculi substrate. Reactions were prepared in triplicate using 0–125 nM of purified protein per reaction. The plate was covered with a lid and incubated at 25°C with shaking. Absorbance (OD600) was recorded at 20-min intervals for 20 hr using a SpectraMax Plus 384 microplate spectrophotometer (Molecular Devices). Hydrolysis of sacculi was monitored over time as a decrease in absorbance.
10
SHP2 Catalytic Activity Assay
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PTP activity was assayed using pNPP
as a substrate at 25 °C in pH 7.0 assay buffer (50 mM 3,3-dimethylglutarate,
1 mM DTT, 1 mM EDTA, and 150 mM NaCl). The assays were performed in
96-well plates in a total reaction volume of 200 μL. The reaction
was initiated by the addition of enzyme (wild-type SHP2 or the LS
SHP2 mutant catalytic domain) to a reaction mixture containing pNPP and the isolated SHP2 N-SH2 domain. For the determination
of Ki, the pNPP concentration
was varied at three different concentrations of the N-SH2 domain.
The reaction rate was measured using a SpectraMax Plus 384 microplate
spectrophotometer (Molecular Devices). The Ki values were determined by fitting the data using the EnzymeKinetics
module in SigmaPlot.
as a substrate at 25 °C in pH 7.0 assay buffer (50 mM 3,3-dimethylglutarate,
1 mM DTT, 1 mM EDTA, and 150 mM NaCl). The assays were performed in
96-well plates in a total reaction volume of 200 μL. The reaction
was initiated by the addition of enzyme (wild-type SHP2 or the LS
SHP2 mutant catalytic domain) to a reaction mixture containing pNPP and the isolated SHP2 N-SH2 domain. For the determination
of Ki, the pNPP concentration
was varied at three different concentrations of the N-SH2 domain.
The reaction rate was measured using a SpectraMax Plus 384 microplate
spectrophotometer (Molecular Devices). The Ki values were determined by fitting the data using the EnzymeKinetics
module in SigmaPlot.
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