All fluorescence measurements were carried using a microplate reader at 30 °C. Binding of (±)-2-methyl-3-ketopentanoyl-SNAC (5 ) to wild-type or mutant, redox-inactive KR0 domains was monitored by following the quenching of protein fluorescence intensity, as previously described.16b (link),34 (link) Successive 5-μL portions of (±)-2-methyl-3-ketopentanoyl-SNAC solution (5 ) were added to 96-well plates (UV-Star® 96-Well Microplates, Greiner Bio-One, black) containing 95 μL of 1 μM protein solution or buffer alone. The final concentration of 5 ranged from 0 to 50 mM (0, 0.2, 0.5, 1.5, 2, 2.5, 3, 3.5, 4, 15, 20, 25, 35, 40, 45, 50 mM). The plate was incubated for 20 min at room temperature. The protein fluorescence was excited at 280 nm (5 nm bandwidth) and monitored at 338 nm (20 nm bandwidth). From the measured fluorescence intensity F at a given final concentration of 5 , the quenched fluorescence intensity Fq was calculated by using Fq=Fapo−Fobs, where the Fapo represents the fluorescence intensity of the apoenzyme in the absence of 5 , and Fobs the fluorescence intensity of apoenzyme at a given concentration of 5 . The binding affinity (KD) was calculated by fitting the observed quenched fluorescence intensity and substrate concentration data to the Ligand Binding Equation (one site saturation, Eq 1 ) using the SigmaPlot 12.5 program (Table S8 ).
Uv star 96 well microplate
Manufactured by Greiner
Sourced in Germany
The UV-Star 96-well microplate is a laboratory equipment designed for UV spectrophotometric analysis. It provides a standardized platform for performing absorbance measurements of samples in a 96-well format.
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Lab products found in correlation
Digital sonifier, by Emerson (2 mentions)
Synergy h1, by Agilent Technologies (2 mentions)
Pirenzepine dihydrochloride, by Merck Group (1 mentions)
Atropine sulphate, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Infinite f200 pro, by Tecan (1 mentions)
Infinite 200 microplate reader, by Tecan (1 mentions)
Infinite f200 pro plate reader, by Tecan (1 mentions)
Epoch 2 microplate reader, by Agilent Technologies (1 mentions)
Clariostar, by BMG Labtech (1 mentions)
Infinite 200 pro multimode reader, by Tecan (1 mentions)
Polycarbonate etched membrane, by Cytiva (1 mentions)
Dulbecco s phosphate buffer saline pbs, by Merck Group (1 mentions)
Eon plate reader, by Agilent Technologies (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Powerwave ht microplate reader, by Agilent Technologies (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Eos 700d, by Canon (1 mentions)
Spectramax m5 plate reader, by Molecular Devices (1 mentions)
20 protocols using uv star 96 well microplate
1
Fluorescence-Based Binding Assay for Ketone Substrate
2
Atropine and Pirenzepine Assay Protocol
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Atropine sulphate, pirenzepine dihydrochloride and 10× phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich Co. (Oakville, ON, Canada). UV-Star 96-well microplates, from Greiner Bio-One, and lens paper were purchased from VWR (Mississauga, ON, Canada). All reagents and materials were used as received.
3
Michaelis-Menten Kinetics of Engineered PAL Variants
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Example 4
Michaelis-Menten graphs with rate V (μM TCA/min) as a function of Phe concentration [Phe] (mM) were generated for wild type PAL3, mPAL1, mPAL2, and mPAL3. Bacteria were inoculated 1:100 from a saturated overnight pre-culture, followed by induction with 200 ng/mL ATC two hours later. After four hours of induction, cells were pelleted, washed in PBS, normalized to OD600=50 in PBS, and diluted 2-fold into 50% glycerol for storage at −80° C. Lysate from each strain was prepared via sonication using a Branson Digital Sonifier with microtip. The soluble fraction of the lysate samples was used for the kinetic assay. Total protein in the lysate samples was measured via Bradford Assay, and all samples were normalized to 10 μg total protein loading per well for the kinetic assay. The lysate samples were incubated in 1×M9 0.5% glucose with Phe concentrations ranging from 40 mM Phe down to 39 μM with 2-fold dilutions. The kinetic assay was performed in UV-star 96-well microplates (Greiner) with TCA quantified by A290 measurements every minute using a BioTek Synergy H1 microplate reader set to 37° C. static incubation. The data points on each graph are rate (V in μM TCA/min) calculated from the first hour of activity for each Phe concentration tested, where activity remained linear. (
4
Spectroscopic Analysis of Antibody-Hemin Interactions
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We used high-throughput absorbance spectroscopy to elucidate the interaction of the repertoire of therapeutic antibodies with hemin in solution. The recombinant antibodies were first diluted to 200 μg/ml in PBS. The dilutions were performed directly in UV-star 96-well microplates (Greiner Bio-One, Les Ulis, France). To 100 μl of antibody solution, an equal volume of freshly prepared hemin solution (10 μM in PBS) was added and intensively homogenized. The resulting concentration of antibodies was 100 μg/ml (670 nM) and of heme was 5 μM. As control heme was added to buffer only. The plates were incubated for 30 min at RT in dark. The absorbance spectra in the wavelength range between 350 and 450 nm were recorded by using Tecan Infinite 200 Pro, the microplate reader. The spectral resolution was 2 nm. All measurements were done at RT. To assess the changes in spectral properties of heme upon interaction with antibodies, the shift in maximal absorbance wavelength was defined as follows: λ-shift = λmax of hemin alone—λmax of hemin in the presence of antibody. The differential spectra (absorbance spectrum of hemin in the presence of antibody—absorbance spectrum of hemin alone) allowed quantification of the maximal increase in the absorbance intensity (ΔA).
5
Kinetic characterization of HSDH
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BT_1911 kinetics were determined with slight modifications to previously established methods[47 (link)]. 0.2 nM HSDH was added to pre-warmed 37°C 100 μL reactions containing 5 mM NAD+ in 10 mM MOPS. Bile acid substrates were included in concentrations ranging from 40 μM to 4.5 mM. Change in absorbance at 340 nm was monitored every 80 seconds in flat bottom UV-Star 96 well microplates (Greiner) from a Tecan Infinite F200 Pro plate reader. A standard curve of NADH was included and reaction absorbances were converted to substrate concentration since NADH is generated stoichiometrically with reaction products. Initial velocity data was plotted and Michaelis-Menten parameters were calculated in GraphPad Prism by fitting the data to a nonlinear regression model.
6
Kinetic Assay for Phenylalanine
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Activated biomass was prepared from 10 mL culture tubes as described in the section above, and then lysed for kinetic parameter determination. To prepare lysate, thawed biomass samples were diluted and sonicated using a Branson Digital Sonifier with microtip, then the soluble fraction of the lysate samples were used for the kinetic assay. Total protein in the lysate samples was measured via Bradford Assay, and all samples were normalized to 10 µg total protein loading per well for the kinetic assay. The lysate samples were incubated in M9 0.5% glucose with Phe concentrations ranging from 40 mM Phe down to 39 μM with twofold dilutions (assay buffer without Phe was also included as a control). The kinetic assay was performed in UV-star 96-well microplates (Greiner) with TCA quantified by A290 measurements every minute using a BioTek Synergy H1 microplate reader set to 37 °C static incubation. Michaelis–Menten model fitting was performed using a nonlinear regression (nls function in R with formula V = (Vmax * [S])/(KM + [S])) for the rate data from three batch replicates. Example model fits can be found in Supplementary Fig. 7 . The rate V used in the nonlinear regression was calculated from the first hour of activity for each Phe concentration tested, where activity remained linear.
7
Kinetic Characterization of PhzF Enzyme
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Enzyme kinetic parameters were determined in an Infinite® 200 microplate reader (Tecan Group AG, Männedorf, Switzerland) with UV-Star® 96-Well microplates (Greiner Bio-One International GmbH, Kremsmünster, Austria) at 25 °C. After determination of a suitable pH value (Fig. S3A ), 40 nM PhzF dimer were added to obtain 100 µl assay solution consisting of 50 mM sodium phosphate pH 7.5, 1% (v/v) DMSO and up to 1 mM DHHA (3 ). Substrate depletion was followed at 275 nm for 20 minutes after reaching a linear phase (Fig. S3B ), using an experimentally determined extinction coefficient of 6500 M−1 cm−1 for DHHA (3 ). The enzyme concentration was increased to 200 and 1000 nM to obtain measurable rates for C3-deuterated DHHA (d-3 ) and for racemic O-Et-DHHA (20 ), respectively. All experiments were performed in triplicate and enzyme kinetic parameters were derived by fitting to a Michaelis-Menten model in GraFit5 (Erithacus Software Ltd., Horley, UK).
8
Kinetic Analysis of HSDH Bile Acid Metabolism
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BT_1911 kinetics were determined with slight modifications to previously established methods (48 (link)). 0.2 nM HSDH was added to pre-warmed 37°C 100 µL reactions containing 5 mM NAD+ in 10 mM MOPS. Bile acid substrates were included in concentrations ranging from 40 µM to 4.5 mM. Change in absorbance at 340 nm was monitored every 80 seconds in flat bottom UV-Star 96-well microplates (Greiner) from a Tecan Infinite F200 Pro Plate Reader. A standard curve of NADH was included and reaction absorbances were converted to substrate concentration since NADH is generated stoichiometrically with reaction products. Initial velocity data were plotted, and Michaelis-Menten parameters were calculated in GraphPad Prism by fitting the data to a nonlinear regression model.
9
UV-Vis Absorption Spectroscopy of Protein Samples
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The UV-Vis absorption were determined according to procedure Rawel et al. [18 (link)] with slight modifications. Protein samples were dissolved in 100 mM sodium phosphate buffer pH 7 (0.1 mg of protein/mL) and 250 µL of each sample was transferred into UV-Star 96-well microplate (Greiner Bio-One, Frickenhausen, Germany). The UV-Vis spectra were measured at the range of 200 to 600 at 20 °C using EPOCH 2 microplate reader (BioTek, Winooski, VT, USA).
10
Ascorbate Oxidation Assay for ROS
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Using a CLARIOstar (BMG LABTECH) plate reader, experiments were carried out in a UV-Star® 96 well microplate (Greiner), Half Area, clear. The absorbance of 100 μM ascorbate (DO ≈ 1.5) at 265 nm was followed under different conditions to indirectly follow ROS production. The final volume per well was 100 μL and was buffered at pH 7.4 using 100 mM HEPES.
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