The inhibition of 3CLpro was determined according to reported protocols with modifications.44 (link) The inhibitor compounds were prepared as stock solutions in DMSO and diluted hundredfold with HEPES buffer (50 mM HEPES, pH 7.5, 0.1 mg mL−1 bovine serum albumin, 0.1% Triton-X100, 0.1 mM DTT) to micromolar concentrations. Volumes of 50 μL of 200 nM MBP-tag SARS-CoV-2 3CLpro (Bioscience) in HEPES buffer or blank HEPES buffer (negative control) were added to the wells of a black 96-well microtiter plate (Greiner Bio One). Volumes of 50 μL of the inhibitor solutions or 1% DMSO in HEPES buffer (positive control) were added. The resulting solutions (100 nM SARS-CoV-2 3CLpro, 0.5% DMSO, 100 μm test compound or blank HEPES buffer) were mixed and incubated at 37 °C for one hour. A volume of 100 μL of 100 μM DABCYL-KTSAVLQSGFRKME-EDANS (Bachem) was added to all wells. The resulting solutions were mixed and the fluorescence emission was measured immediately every 3 min for 75 min (λexc = 355 nm; λem = 460 nm) at 37 °C using a Victor™ X4 Perkin Elmer 2030 multilabel reader. The increase of emission over time followed a linear trend (r2 >0.97) and the enzymatic activities were calculated as the slope thereof. The wells containing the negative control were used to confirm the absence of false positive results by reaction of the inhibitor compound with the fluorogenic substrate.
Black 96 well microtiter plate
Manufactured by Greiner
Sourced in Germany, Austria, United States
The Black 96-well microtiter plate is a laboratory equipment item designed for various assay applications. It features 96 individual wells in a standard microplate format, with a black-colored surface that helps reduce background fluorescence and improve contrast for optical measurements.
Automatically generated - may contain errors
Lab products found in correlation
Sars cov 2 plpro, by Elabscience (2 mentions)
Dabcyl ktsavlq sgfrkme edans, by Bachem (1 mentions)
Victor x4, by PerkinElmer (1 mentions)
Trypsin, by AppliChem (1 mentions)
Ni nta tips, by Molecular Devices (1 mentions)
Dip and read biosensors, by Molecular Devices (1 mentions)
Octet red96 system, by Molecular Devices (1 mentions)
Infinite f200 microplate reader, by Tecan (1 mentions)
Fluozin 3, by Thermo Fisher Scientific (1 mentions)
Z arg leu arg gly gly amc, by Bachem (1 mentions)
Boc lys tfa amc, by Bachem (1 mentions)
Pherastar optima, by BMG Labtech (1 mentions)
Fluostar omega spectrophotometer, by BMG Labtech (1 mentions)
Fluostar optima, by BMG Labtech (1 mentions)
Infinite m200, by Tecan (1 mentions)
Fluo 4 nw calcium assay kit, by Thermo Fisher Scientific (1 mentions)
Nanoquant plate, by Tecan (1 mentions)
Spectramax m2 microplate reader, by Molecular Devices (1 mentions)
96 well polystyrene microtiter plate, by Greiner (1 mentions)
Synergy h1 multi mode reader, by Agilent Technologies (1 mentions)
17 protocols using black 96 well microtiter plate
1
SARS-CoV-2 3CL Protease Inhibition Assay
2
HDAC4 Enzyme Inhibition Assay
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A serial dilution of TZD in assay buffer (25 mM TRIS-HCl, 75 mM KCl, 0.001% Pluronic F-127, pH 8.0) was incubated with 1 nM cdHDAC4wt in a black 96-well microtiter plate (Greiner). Afterwards the enzymatic reaction was initiated by the addition of 20 µM Boc-Lys{TFA}-7-Amino-4-methylcoumarin (Bachem, Bubendorf, Switzerland) as substrate. After incubation the enzymatic reaction was terminated by the addition of 1.7 µM SATFMK. The deacetylated substrate was converted into a fluorescent product by the addition of 0.4 mg/mL trypsin (AppliChem, Darmstadt, Germany). The release of fluorogenic substrate was followed in a microplate reader (PherStar Plus, BMG Labtech, Ortenberg, Germany) at 450 nm (λEx = 350 nm) and correlated to enzyme activity. GraphPad Prism program was used to generate dose response curves and were fitted to a four parameter logistic function to obtain IC50-values [33 (link)]:
in which EA is the enzyme activity for a given inhibitor concentration x, Emax and E0 are the enzyme activities in the absence of inhibitor and at complete inhibition, respectively. h is the slope of the curve and IC50 is the inhibitor concentration at which half of the enzyme activity is inhibited. All incubations steps were performed for 60 min at 30 °C and 450 rpm.
in which EA is the enzyme activity for a given inhibitor concentration x, Emax and E0 are the enzyme activities in the absence of inhibitor and at complete inhibition, respectively. h is the slope of the curve and IC50 is the inhibitor concentration at which half of the enzyme activity is inhibited. All incubations steps were performed for 60 min at 30 °C and 450 rpm.
3
Disulfide Bond Effects on HDAC8 Activity
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For the determination of disulfide bond induced changes in enzyme activity 20 nm of the prior treated HDAC8 dilution was mixed with 20 μm Boc‐Lys(trifluoroacetyl)‐AMC as substrate in a black 96‐well microtiter plate (Greiner) in assay buffer for 15 min at 30 °C. For the HDAC8C102S/C153S mutant the substrate reaction was conducted for 1 h. After substrate conversion the reaction was stopped by the addition of 1.7 μm SATFMK and fluorescent AMC was released by the addition 0.4 mg mL−1 trypsin. Relative fluorescent units were measured in a microplate reader at 450 nm (λex=350 nm) and normalized to the untreated control.
4
Measuring αIFNα-ab Dissociation Kinetics
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The dissociation constant of αIFNα-ab was measured by biolayer interferometry with the Octet Red96 system (fortéBio, Menlo Park, CA, USA) using Dip and Read Biosensors (fortéBio) [47 ]. Kinetic measurements were performed in kinetic buffer (0.2% BSA in PBST, PBST was PBS with 0.05% [v/v] Tween 20). Kinetic buffer without αIFNα-ab was applied to Ni-NTA tips (fortéBio) for 300 sec. Then, αIFNα-ab was coupled in two steps to the Ni-NTA tips. Firstly, the tip was activated for 80 s in 0.1 M EDC, 0.0025 M NHS for covalent coupling and secondly placed for 600 s in a solution of 10 μg/mL αIFNα-ab in kinetic buffer. After covalent binding of αIFNα-ab to the tip, free amino-groups were saturated with 1 M ethanolamine, pH 8.5 for 60 s, followed by equilibration with kinetic buffer for 600 s and recording a baseline for 60 s in wells of a black 96 well microtiter plate (Greiner Bio-One, Austria) to allow subtraction of a baseline drift resulting from unspecific binding of BSA. Tips loaded with αIFNα-ab were then dipped in parallel in 200 μl of increasing concentrations of IFNα in kinetic buffer. The biosensor tips were finally transferred into kinetic buffer to measure the dissociation of αIFNα-ab. Orbital shake speed was 1000 rpm. Fit curves were calculated with the Octet Data Acquisition program 8.2.0.9.
5
Sensitive Virus Detection by Sandwich FLISA
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Sandwich FLISA was performed as described previously [29 (link)]. Briefly, a black 96-well microtiter plate (Greiner GmbH) was coated with 100 µL/well of 10 µg/mL antibody candidate in coating buffer (pH 9.6) and incubated at 4 °C for 12 h. After 5% non-fat dry milk blocking at 37 °C for 2 h, the recombinant virus (100 µL/well) was added and the plate was incubated for another hour at 37 °C. After washing the plate with 200 μL PBS-T (pH 7.4), Eu nanoparticle (NP)-conjugated antibody (100 μL/well) was added and the plate was incubated at 37 °C for another hour for antigen detection. Stringent washing with PBS-T (pH 7.4) was performed five times to remove nonspecific binding, and 100 μL PBS was added to each well. Fluorescence was then measured in the Infinite F200 microplate reader (TECAN, Männedorf, Switzerland) at 355 nm (excitation) and 612 nm (emission) wavelengths.
6
Zinc-ejecting Inhibitor Assay for SARS-CoV-2 PL^pro
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To determine if the inhibitors are Zn-ejecting agents, the presence of free Zn2+ in solution was measured as previously described.11 (link) The inhibitor compounds were prepared as stock solutions in DMSO and diluted hundredfold with HEPES buffer (50 mM HEPES, pH 7.5) to 2 μM and 20 μM concentrations. Volumes of 50 μL of 1 μM SARS-CoV-2 PLpro (Elabscience) in HEPES buffer or blank HEPES buffer (negative control) were added to the wells of a black 96-well microtiter plate (Greiner Bio One). Volumes of 50 μL of the inhibitor solutions or 1% DMSO in HEPES buffer (positive control) were added. The resulting solutions (500 nM PLpro SARS-CoV-2, 0.5% DMSO, 1 μM or 10 μM test compound or blank HEPES buffer) were mixed. A volume of 100 μL of 2.0 μM of zinc-specific fluorophore FluoZinTM-3 (Invitrogen/Life Technologies) was added to all wells. The resulting solutions were mixed and the fluorescence emission was measured immediately every 5 min for 100 min (λexc = 485 nm; λem = 535 nm) at 37 °C using a Victor™ X4 Pekin Elmer 2030 multilabel reader. The wells containing the negative control were used to confirm the absence of false positive results by reaction of the inhibitor compound with the zinc-specific fluorophore.
7
Inhibition Kinetics of SARS-CoV-2 PLpro Protease
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The inhibitor compounds were prepared as stock solutions in DMSO and diluted hundredfold with HEPES buffer (50 mm HEPES, pH 7.5, 0.1 mg mL−1 bovine serum albumin, 0.1% Triton-X100) to micromolar concentrations. Volumes of 50 μL of 200 nM SARS-CoV-2 PLpro (Elabscience) in HEPES buffer were added to the wells of a black 96-well microtiter plate (Greiner Bio One). Volumes of 50 μL of the inhibitor solutions or 1% DMSO in HEPES buffer (positive control) were added. The resulting solutions (100 nm SARS-CoV-2 PLpro 0.5% DMSO, 0.1–0.75 μm test compound or blank HEPES buffer) were mixed and incubated at 37 °C for 10 min. A volume of 100 μL of 20–8000 μm of substrate (Z-Arg-Leu-Arg-Gly-Gly-AMC, Bachem) was added to all wells. The resulting solutions were mixed and the fluorescence emission was measured immediately every minute for 1 h (λexc = 355 nm; λem = 460 nm) at 37 °C using a Victor™ X4 Perkin Elmer 2030 multilabel reader. The enzyme activity of PLpro was represented by the Michaelis Menten equation (eqn (1)) and the Km and Vmax values were calculated with Lineweaver–Burk equation (eqn (2)) where the slope is Km/Vmax, the intersection with the Y-axis corresponds to 1/Vmax, and intersection with the X-axis corresponds to −1/Km.
8
Quantifying Pneumococcal Cell Wall Binding
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Five mg of cell wall pneumococcal sacculi were resuspended with 50 μl of each FITC-labelled proteins at 1 mg/ml in 10 mM HEPES, 150 mM NaCl, 2 mM CaCl2 (pH 7.4) and incubated for 1 h at RT. Sacculi were centrifuged (5 min at 20,000 g), supernatants were removed and the pellets were resuspended in 1 mL of 10 mM HEPES, 150 mM NaCl, 2 mM CaCl2 (pH 7.4). This washing step was repeated five times. The sacculi pellets were finally resuspended in 50 μL of 10 mM HEPES, 150 mM NaCl, 2 mM CaCl2 (pH 7.4) and transferred into a black 96-well microtiter plate (Greiner Bio One). Bound proteins to pneumococcal sacculi were detected by fluorescence measurements (Fluostar Optima; BMG). Each experiment was performed in duplicate.
9
Kinetic Characterization of KDAC8 Enzyme
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The measurements were carried out in a black 96-well microtiter plate (Greiner) at 30°C in assay buffer (see above). A serial two-fold dilution ranging from 50 µmol/L substrate, Boc-Lys(TFA)-AMC (Bachem), to 780 nmol/L, was prepared in an assay buffer containing 0.1 mg/mL trypsin. The enzymatic reaction was initiated by the addition of 10 nmol/L KDAC8. The release of fluorogenic AMC was observed in a micro plate reader (PHERAstar Optima, BMG Labtech) at 450 nm (Ex: 350 nm). The data points were plotted against time and blank corrected. For the determination of Michaelis-Menten parameters, a linear regression analysis of the initial slope (vi) of each curve was performed. Slopes were transformed into rates of AMC product formation using a calibration curve with different AMC concentrations. The obtained enzyme conversion rates were plotted against the respective substrate concentration and fitted to the Michaelis-Menten function using GraphPad Prism software. The turnover number (kcat) was calculated as
where vi is the substrate conversion rate and E is the enzyme concentration. All solutions were pre-incubated at 30°C.
where vi is the substrate conversion rate and E is the enzyme concentration. All solutions were pre-incubated at 30°C.
10
Fluorogenic Assay for Protease Activity
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A fluorogenic protease assay was used to measure the proteolytic activity of BAJ93208. The quenched fluorogenic peptide (5-amino-2-nitrobenzoyl-S-D-Y-P-K-L-N-L-L-P-K-7-methoxycoumarinyl-4-acetyl) was synthesized by JPT Peptide Technologies GmbH (Berlin, Germany). In a standard reaction, 10 μl of protease containing sample was added to a solution containing a final concentration of 2 μM fluorogenic peptide, 25 mM MES, pH 6.5, supplemented with 150 mM NaCl and 10 mM CaCl2. Kinetic assays were performed at 25 °C and formation of the MCA – labeled cleavage product was followed at 330 nm excitation and 420 nm emission using black 96 well microtiter plates (Greiner, Germany) in a FLUOstar Omega spectrophotometer (BMG Labtech).
Specific activity was determined using 10 μM fluorogenic peptide and 0.1 μg of enzyme in 25 mM MES, pH 6.5 in a 200 μl reaction with or without 5 mM DTT. Serial dilutions of 7-amino-4-methylcoumarin (AMC, Peptanova, Germany) ranging from 0 to 150 pM per well in 25 mM MES at pH 6.5 were prepared and relative florescence units per pM free MCA were measured. The value was corrected by the difference between background fluorescence and expected maximal fluorescence and used to calculate the specific activity of the protease.
Specific activity was determined using 10 μM fluorogenic peptide and 0.1 μg of enzyme in 25 mM MES, pH 6.5 in a 200 μl reaction with or without 5 mM DTT. Serial dilutions of 7-amino-4-methylcoumarin (AMC, Peptanova, Germany) ranging from 0 to 150 pM per well in 25 mM MES at pH 6.5 were prepared and relative florescence units per pM free MCA were measured. The value was corrected by the difference between background fluorescence and expected maximal fluorescence and used to calculate the specific activity of the protease.
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