Inhibition concentrations were determined using a FRET-based diUb cleavage reaction to measure isopeptide hydrolysis by the STAMBPJAMM domain. Fluorescence of hydrolyzed K63-linked diUb (TAMRA/QXL position 3 labeled, Boston Biochem #UF-330) was measured on a BioTek synergy LX plate reader equipped with a red filter cube assembly (ex. 530 nm, em. 590 nm) for 60 min at ambient temperature (22 °C). Enzyme (2 nM) and UbVs were preincubated for 10 min at ambient temperature before addition of the diUb substrate (100 nM) in 50 μl reaction buffer (50 mM HEPES pH7.5, 100 mM NaCl, 0.01% v/v Tween 20, 0.01 mg/ml BSA, 5 mM DTT). Reactions were replicated in triplicate (N = 3) for varying concentrations of UbV (1 μM–0.1 nM). The rate of reaction was determined from the slope of the initial linear phase of the reaction. Reaction rates were averaged and normalized to the negative control (no inhibitor) then plotted against UbV concentrations in GraphPad Prism 8. In total, 50% inhibition concentrations were determined by nonlinear regression using the formula for inhibitor concentration versus normalized response (variable slope).
Synergy lx plate reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy LX plate reader is a versatile instrument designed for various life science and biochemistry applications. It is capable of performing absorbance, fluorescence, and luminescence measurements on microplates. The Synergy LX provides accurate and reliable data for a wide range of assays, including enzyme-linked immunosorbent assays (ELISAs), cell-based assays, and more.
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6 protocols using synergy lx plate reader
1
STAMBP JAMM Domain Inhibition Assay
2
Quantifying Oxidative Stress in Insect Tissues
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Oxidative stress levels were examined using a non-enzymatic component of the antioxidant system, glutathione (GSH) and the ratio of GSH to oxidized glutathione (GSSG). GSH/GSSG ratio and the amount of GSH and GSSG were examined in the thorax tissue of queens using a spectrophotometric GSH/GSSG assay kit (Eagle Biosciences). Thoraxes were separated from the rest of the body and weighed prior to analysis. To prevent oxidation, the thorax tissue was ground on ice in a glass tissue grinder with 1 ml 7.4100 mM Tris–HCL buffer, following a modified protocol by (Lalouette et al., 2011 (link)). The homogenate was centrifuged at 4°C for 15 min until the supernatant was clear. Glutathione (GSH) and oxidized glutathione (GSSG) were measured in triplicate and measurements were taken every minute for 10 min at 412 nm using a BioTek Synergy LX PlateReader. GSH and GSSG contents were calculated from standard curves using the same kinetic measurement approach, and the ratio of GSH/GSSG was calculated using the following equation (GSH-2*GSSG)/GSSG) according to the manufacturer’s instructions.
3
Cell Surface ELISA for Viral Glycoproteins
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Cell surface ELISA was performed as previously described40 (link) with several modifications. 96w plates with transfected cells were washed 3x with PBS supplemented with divalent cations (0.9 mM CaCl2, 0.5 mM MgCl2 (PBS2+)) 20 h post-transfection and fixed with 4% PFA in PBS for 10 min followed by 3x washes and blocking in 3% BSA in PBS2+ for 30 min. The cells were then incubated with mouse anti-gB (1:20) or anti-gD (1:100) mAbs in blocking buffer for 1 h at room temperature, followed by 5x washes and incubation with rabbit anti-mouse Igs HRP (1:500) for 45 min and 6x washes. Plates were developed using TMB + substrate chromogen (Thermo Scientific) and stopped with 0.5 M H2SO4. The absorbance at 450 nm was quantified using BioTek Synergy LX plate reader.
4
Anticoagulant Activity Quantification
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Human plasma ATIII (1 μM) (Sigma-Aldrich) in 50 mM tris-HCl, 175 mM NaCl, and 7.5 mM EDTA (pH 8.4) and bovine FXa (1 μM) (Sigma-Aldrich) were both diluted 1:30 in 0.9% NaCl, and 8 mM FXa substrate (Sigma-Aldrich) was diluted 1:10 in 0.9% NaCl immediately before assay. ATIII (37.5 μl) was added to each well of a 96-well plate before adding heparin/HS samples at a range of concentrations diluted to 12.5 μl in 0.9% NaCl. Mixtures were incubated for 2 min at 37°C before addition of bovine FXa (37.5 μl) followed by 1-min incubation at 37°C. The FXa substrate (37.5 μl) was then added followed by incubation at 37°C for 10 min before 37.5 μl of acetic acid was used to stop the enzymatic reaction. Absorbance was read at 405 nm using a Synergy LX plate reader (BioTek), and IC50 values used for quantification of anticoagulant activity relative to PMH were determined with an online AAT Bioquest IC50 calculator.
5
Fungal Growth Kinetics with Algal Extracts
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Fungal growth kinetics with slight modification [17 (link)] was measured using a 96-well plate (Bar-Naor Ltd., Petah Tikva, Israel). Algal ethanolic, acetonic, and ulvan extracts of 1% to 0.125% were diluted in 1% SMB. Then, 200 µL from each dilution was pipetted into a 96-well plate in triplicate. Conidial suspension of 10 µL (105 Conidia mL−1) was added to each well and mixed thoroughly. “Blanks” were filled with algal dilutions without conidial suspension. The plate was incubated at room temperature, and optical density measurements at 600 nm (OD600) were taken every hour for 60 h using a Synergy LX plate reader (BioTek, Winooski, VT, USA). The absorbance of three wells of repeats was averaged together and was background-corrected by subtracting the average absorbance of media alone at time zero [17 (link)].
6
Fluorescence Assay Optimization Protocol
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For initial assay chemistry optimization, a Synergy LX plate reader from BioTek (500 nm excitation, 530 nm emission) was used to measure the fluorescence. After the alkane is melted, 15 μL of the assay liquid was moved to a 384-well plate. A fluorescence measurement was taken after 20 min.
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