Recombinant human ACE2-Fc (Genscript, Piscataway, NJ, USA) at a concentration of 2 μg/mL in a phosphate buffer saline (PBS) was adsorbed to wells of ELISA plates at 4 °C overnight. Serum dilutions were prepared in 1% BSA/PBS-T and incubated with equal amounts of spike protein (the final concentration of the spike protein in all samples was 0.1 μg/mL) for 1 h at 37 °C. After blocking in 1% BSA/PBS-T for 1 h at 37 °C, the plates were washed in PBS-T, and pre-incubated serum-spike samples were added to the wells and incubated for 2 h at room temperature. After washing, the plates were incubated with an HRP conjugated engineered form of streptavidin (streptactin-HRP) (1:) in 1% BSA/PBS-T for 1 h at room temperature. After the final wash, a TMB substrate was added, and the reaction was stopped by the addition of an acid solution (3M H3PO4). Absorbance was measured by the Synergy Mx microtiter plate reader (Biotek, Winooski, VT, USA).
Synergy mx microtiter plate reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy Mx microtiter plate reader is a versatile instrument designed for a wide range of microplate-based assays. It features a monochromator-based optical system that enables the measurement of absorbance, fluorescence, and luminescence in a variety of microplate formats.
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7 protocols using synergy mx microtiter plate reader
1
SARS-CoV-2 Spike Protein Binding Assay
2
Quantifying Msp1 Protein in L. rhamnosus Supernatants
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The protein concentration in the cell-free supernatant of L. rhamnosus GG and CMPG5540 was determined using bicinchoninic acid (BCA) protein assay. The wells of a 96- well ELISA plate (Greiner, Bio-one) were coated overnight with supernatant (0.5 µg/mL), after lyophilization and resolving in PBS, or Msp1 (at different concentration, standard curve) at 37 °C. Afterwards, the wells were washed three times with PBS/T (PBS with 0.05% Tween 20), 250 µL PBS/T with 25% solution of skimmed milk was added, followed by a 1 hour incubation at 37 °C to block aspecific binding. Next, the wells were washed three times with PBS/T and each well was then filled with 100 µL of Msp1 antiserum diluted 1:2000 in PBS/T and incubated (37 °C, 90 min). Alkaline phosphate-conjugated goat anti-rabbit immunoglobulin G (IgG, Sigma) was diluted 1:3000 in PBS/T and added to each well (100 µL) before incubation (37 °C, 1 h). After incubation (30 min, 37 °C) of the bound antibodies with 150 µL of p-nitrophenyl phosphate (1 mg/mL in 1 M Tris-HCl, pH 9.8) (Sigma) per well, the absorbance (405 nm) of each well was read with a Synergy MX microtiter plate reader (Biotek Instruments).
3
SARS-CoV-2 Spike Protein Binding Assay
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Recombinant human ACE2‐Fc protein (Genescript) was absorbed to ELISA plates at 2 µg/mL in 50 µL/well of phosphate buffer saline (PBS) at 4°C overnight. The plates were washed five times using PBS + 0.01% Tween and blocked in 0.45 mM BSA in PBS for 1 hour. Recombinant spike‐STREP tag (0.2 µg/mL) was prepared as described in 21 .The protein was incubated with the indicated concentrations of compounds: NACA (0.5, 2, 5, and 10 mM), auranofin (0.1, 0.2, 0.5, and 1 mM), L‐ascorbic acid (5, 10, and 20 mM), and JTT‐705 (20, 50, and 200 µM) for 1 hour in PBS at 37°C. Then samples were added to wells and incubated for an additional 2 hours at room temperature. After washing, the plate was incubated with STREP‐Tactin‐HRP (1:10 000) in BSA/PBS for 1 hour at room temperature. After the final wash, TMB substrate was added and the reaction was stopped by the addition of 3 M H3PO4. Absorbance was measured using a Synergy Mx microtiter plate reader (Biotek). DMSO and EtOH backgrounds were added to the auranofin and JTT‐705 results.
4
Bacterial Culture and Standardization
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Tryptic soy broth (TSB) was used as the growth medium for both Gram-negative E. coli K12 and Gram-positive B. subtilis strains. From a cultured bacterial stock on a trypticase soy agar (TSA) plate, an isolated colony was picked and inoculated in 20 mL TSB. This was grown overnight at 35 °C with mild shaking. The following day, bacterial cultures were centrifuged at 10,000 rpm for 5 min, and washed 3 to 4 times with sterilized deionized (DI) water. The resulting bacterial pellets were re-suspended in sterilized DI water. Optical density (OD) of the suspensions were adjusted to 0.5 at 600 nm wavelength using a Synergy MX Microtiter plate reader (Synergy 4, Biotek, Winooski, VT, USA). This OD roughly matches to a bacterial concentration of 107 CFU/mL.
5
Coupled Enzyme Assay of XylA Activity
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Activity of XylA was measured with a coupled enzyme assay using d -sorbitol dehydrogenase70 . d -sorbitol dehydrogenase (SDH) was obtained from Roche Diagnostics GmbH (Mannheim, Germany). Reactions were performed at 30 °C and pH 7.0 (20 mM MOPS buffer). The decrease in absorbance at 340 nm was monitored in either a spectrophotometer (Jasco, Easton, MD) or a Synergy Mx microtiter plate reader (BioTek Instruments, Winooski, VT). Reaction mixtures included 5, 200 or 500 mM xylose, 250 μM NADH and U ml−1 of SDH. Addition of 0.03 to 1 μM (depending on the substrate concentration and the metal added) XI or cell free extract into the mixture initiated the reaction. For measuring XylA activity in the presence of different metal cofactors, samples of apo-XylA were prepared by overnight incubation of the purified enzyme with 10 mM EDTA. Subsequently, EDTA was removed by buffer exchange to 20 mM MOPS, pH 7.0 and 1 mM of divalent metal solutions (MgCl2, MnCl2 or CaCl2) were added in the reaction. For kinetic analyses, d -xylose was added at concentrations ranging from 0.5 mM to 1.50 M.
6
Bacterial Growth Optimization Protocol
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Bacterial strains and plasmids used in this study are listed in Table 1 . E. coli cultures were incubated at 37°C. For cloning procedures and during genome engineering manipulations, cells were grown in a lysogeny broth (LB) medium (10 g l−1 tryptone, 5 g l−1 yeast extract and 10 g l−1 NaCl; solid culture media additionally contained 15 g l−1 agar). All cultures were agitated at 200 rpm (MaxQ™ 8000 incubator; ThermoFisher Scientific, Waltham, MA, USA). Streptomycin (Str) was added whenever needed at 100 μg ml−1. The optical density measured at 600 nm (OD600) was recorded in a Genesys 20 spectrophotometer (Thermo Fisher Scientific) to estimate bacterial growth. During physiological characterization of engineered strains, growth kinetics were followed at OD600 with light path correction in a Synergy™ MX microtiter plate reader (BioTek Instruments Inc., Winooski, VT, USA).
7
Fluorescent Biosensor for Intracellular F- Monitoring
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Plasmid pS441·FRSv1 (Calero et al., 2020 ), carrying a monomeric superfolder gfp (msfGFP) gene under control of a synthetic riboswitch‐promoter element, was transformed into both the wild‐type strain and the P. putida deletion mutants constructed in this work and selected according to the Tn‐Seq analysis. The intracellular F− concentration in these strains was correlated to the msfGFP fluorescence output. To this end, overnight pre‐cultures of strains carrying plasmid pS441·FRSv1 were diluted 20 times in 5 ml of M9 minimal medium and the cell suspension was distributed in 96‐well microtiter plates (flat bottom; Greiner Bio‐One, Kremsmünster, Austria). Cells were grown for 3 h at 30°C with agitation at 300 rpm, after which the cultures were added with NaF at 0, 0.25, 1 and 15 mM. The kinetics of bacterial growth (estimated as the OD600) and msfGFP fluorescence, using wavelengths of excitation and emission of 485 and 528 nm, respectively, were followed in a SynergyMX microtiter plate reader (BioTek Instruments Inc.) for 20 h. Measurements were carried out every 10 min on three independent biological replicates, and the normalized fluorescence values at 10 and 15 h after NaF addition were plotted and compared across conditions.
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