100 ng of Human recombinant folate receptor 1 (FOLR1) diluted at 100 ng/25 μL in Tris-buffered saline (TBS; Bio-Rad) and human recombinant integrin α4β1 (all from R&D systems) diluted at 100 ng/25 μL in TBS supplemented with 1 mM MnCl2 was placed in 96-well half-area microplates (Corning) and incubated at 4°C overnight. After 1-h blocking with 3% (v/v) skim milk in TBS, 5 μg/mL of unconjugated h38C2 IgG1 and h38C2 IgG1 chemically programmed with folate (compounds 3 and 5 ) or LLP2A (compounds 4 and 6 ) were added to the FOLR1-or integrin a4D1-coated wells, respectively, and incubated for 1 h. Subsequently, the wells were washed 3 times with 0.05% (v/v) Tween 20 (Sigma-Aldrich) in TBS. A 1:2,000 dilution of HRP-conjugated goat anti-human IgG Fcy-specific pAbs (Jackson ImmunoResearch) in 3% (v/v) skim milk in TBS was added and incubated for 1 h at RT. Following 3 washes as before, BioFX ABTS One Component HRP Microwell Substrate (Surmodics) was added to the wells following the manufacturer’s instructions. Absorbance at 405 nm was detected using a Spectra Max M5 instrument. The experiment was performed in triplicate.
96 well half area microplate
Manufactured by Corning
Sourced in United States
The 96-well half-area microplates are a versatile laboratory equipment designed for a variety of applications. These microplates feature a reduced well volume compared to standard 96-well plates, making them suitable for experiments or assays that require smaller sample sizes. The half-area format allows for efficient utilization of reagents and samples, while maintaining the standard 96-well layout for compatibility with various laboratory equipment and automation systems.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax 190 microplate reader, by Molecular Devices (4 mentions)
Tmb substrate solution, by Merck Group (3 mentions)
Horseradish peroxidase hrp conjugated anti m13 antibody, by Sino Biological (2 mentions)
Safiremicroplate spectrophotometer, by Tecan (2 mentions)
Tween 20, by Merck Group (1 mentions)
Tetramethylbenzidine tmb substrate solution, by Merck Group (1 mentions)
Enzcheck phosphate assay kit, by Thermo Fisher Scientific (1 mentions)
Multiskan go plate reader, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
1 n sulfuric acid, by Merck Group (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Hrp conjugated anti m13 antibody, by Sino Biological (1 mentions)
Infinite m200, by Tecan (1 mentions)
Ethyl acetoacetate, by Merck Group (1 mentions)
3 pentanone, by Merck Group (1 mentions)
Cyclohexanone, by Merck Group (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Xylene, by Merck Group (1 mentions)
Glass vial, by Thermo Fisher Scientific (1 mentions)
15 protocols using 96 well half area microplate
1
Folate Receptor and Integrin Binding Assay
2
Monoclonal Phage ELISA for MERS-CoV Spike Protein
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Monoclonal phage ELISA was performed after three rounds of panning. Several 96-Well Half-Area Microplates (Corning, New York, NY, USA) were coated overnight at 4 °C with 30 μL per well of 1 μg/mL MERS-S2P and each well was blocked with 5% skim milk in PBS for 1 h at RT. The amplified phages of 94 individual clones from the third round of panning were added and incubated for 1 h at 37 °C. After washing four times with PBS-T, horseradish peroxidase (HRP)-conjugated anti-M13 antibody (1:5000, Sino Biological, Beijing, China) was incubated for 1 h at 37 °C. After washing four times with PBS-T, 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate solution (Sigma-Aldrich) was added for 8 min, and the reaction was stopped with 1 N sulfuric acid (Merck, Darmstadt, Germany). Absorbance was measured at 450 nm using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnydale, CA, USA). To demonstrate MERS-S2P specificity, monoclonal phage ELISA was performed with a SARS-CoV spike protein (Abcam, Cambridge, U.K.) and human coronavirus HKU1 spike protein (Sino Biological) as controls.
3
Quantifying Ras GTPase Activity
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GST-fused KRas (WT and G12D mutant) was loaded with GTP (Sigma-Aldrich, G8877) following the protocol for the preparation of GppNHp-loaded Ras protein. GTPase activity of Ras proteins was measured using an EnzCheck phosphate assay kit (Thermo Scientific, E6646) according to the manufacturer's instructions27 (link). Briefly, assay buffer only (intrinsic GTPase activity), indicated antibody (15 μM final concentration) or RasGAP (15 μM final concentration) was added to a reaction mixture (30 μM GTP-loaded KRas protein (WT or G12D), 200 μM 2-amino-6-mercapto-7-methylpurine riboside (MESG), and 50 U ml–1 purine nucleoside phosphorylase (PNP) in assay buffer (50 mM Tris-HCl, pH 8.0, 1 mM DTT)) in 96-well half-area microplates (Corning). GTP hydrolysis was initiated by the addition of MgCl2 (10 mM final concentration). The absorbance at 360 nm was measured at 20 °C for 20 min at every 10 s interval on a MULTISKAN GO plate reader (Thermo Scientific). The phosphate concentration ([Pi]t) at each time point was determined by comparison with a phosphate standard curve and plotted against time. The hydrolysis rate constant (k) was determined by fitting the data to a single-phase, exponential non-linear regression curve with the equation [Pi]t=[Pi]0+([Pi]final−[Pi]0) (1−exp(−kt)) in GraphPad Prism software27 (link).
4
Arf-Dependent GEF Activity Assay
5
Arf-Dependent GEF Activity Assay
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Grp1 and Grp1-Arf6 fusion constructs with or without 80 μM
Arf6NΔ13-GppNHp or 10 μM IP4 were formatted into
96 well half area microplates (Corning) and incubated for 16–24 hrs
at 25°C in 20 mM Tris, pH 8.0, 150 mM NaCl, 2 mM MgCl2and 250 μM GppNHp. Exchange reactions were initiated by addition of
1 μM Arf1NΔ17-mantGDP and monitored using a Safire
microplate spectrophotometer (Tecan) with excitation at 360 nm and emission
at 440 nm. Observed rate constants (kobs) were obtained by
fitting with It = (I0 −
I∞) exp(−kobs t) +
I∞, where It, I0, and
I∞ are the emission intensities at times t, t
= 0, and t→∞. Catalytic efficiencies
(kcat/KM) were obtained from the slope of a linear
least squares fit with kobs =
(kcat/KM) [GEF] +
kintr, where kintr is the intrinsic rate
constant.
Arf6NΔ13-GppNHp or 10 μM IP4 were formatted into
96 well half area microplates (Corning) and incubated for 16–24 hrs
at 25°C in 20 mM Tris, pH 8.0, 150 mM NaCl, 2 mM MgCl2and 250 μM GppNHp. Exchange reactions were initiated by addition of
1 μM Arf1NΔ17-mantGDP and monitored using a Safire
microplate spectrophotometer (Tecan) with excitation at 360 nm and emission
at 440 nm. Observed rate constants (kobs) were obtained by
fitting with It = (I0 −
I∞) exp(−kobs t) +
I∞, where It, I0, and
I∞ are the emission intensities at times t, t
= 0, and t→∞. Catalytic efficiencies
(kcat/KM) were obtained from the slope of a linear
least squares fit with kobs =
(kcat/KM) [GEF] +
kintr, where kintr is the intrinsic rate
constant.
6
Screening for SARS-CoV-2 RBD Binders
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Individual phage clones from either the third or fourth round were tested for binding to the SARS-2 RBD-coated plate. Several 96-Well Half-Area Microplates (Corning, Cat. 3690, New York, NY, USA) were coated overnight at 4 °C, with 30 μL per well of 1 μg/mL SARS-2 RBD, and each well was blocked with 5% skimmed milk in PBS for 1 h at room temperature. The amplified phages of individual clones from the third or fourth rounds of panning were added and incubated for 1 h at 37 °C. After washing four times with PBST, the horseradish peroxidase-conjugated anti-M13 antibody (1:5000, Sino Biological, Cat. 11973-MM05, Beijing, China) was incubated for 1 h at 37 °C. After washing four times with PBST, a TMB substrate solution (Sigma-Aldrich, Cat. T0440, St. Louis, MO, USA) was added for 8 min, and the reaction was stopped with 1 N sulfuric acid (Merck, Cat. 100731, Darmstadt, Germany). The absorbance was measured at 450 nm using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnydale, CA, USA).
7
ELISA Quantification of Biomarkers
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TNF‐α, PlGF, and sFLt‐1 ELISA kits for humans were purchased from Thermo Fisher (Waltham, MA), Invitrogen (Waltham, MA) and Bio‐Techne (Minneapolis, MN), respectively. Corning® (Corning, NY) 96 well half‐area microplates were used for TNF‐α ELISA analysis. The kits from Invitrogen and Bio‐Techne came with their own coated 96‐well microplates. Total protein was measured by BCA protein assay (Thermo Fisher 23225). The assays were performed according to the manufacturer's protocol. The ELISA concentrations were all normalized to their corresponding total protein concentrations. GraphPad Prism 8.1 software was used to read the absorbance of the well plates.
8
Monoclonal Phage ELISA for YKL-40 Detection
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A monoclonal phage ELISA was performed after three rounds of panning. Several 96-Well Half-Area Microplates (Corning, New York, NY, USA) were coated overnight at 4 °C, with 30 μL per well of 1 μg/mL hYKL-40, and each well was blocked with 5% skim milk in PBS for 1 h at RT. The amplified phages of individual clones from the third round of panning were added and incubated for 1 h at 37 °C. After washing four times with PBS-T, horseradish peroxidase (HRP)-conjugated anti-M13 antibody (1:5000, Sino Biological, Beijing, China) was incubated for 1 h at 37 °C. After washing it four times with PBS-T, a TMB substrate solution (Sigma-Aldrich, St. Louis, MO, USA) was added for 8 min, and the reaction was stopped with 1 N sulfuric acid (Merck, Darmstadt, Germany). The absorbance was measured at 450 nm using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnydale, CA, USA).
9
ELISA for anti-DrSA antibodies
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96-well half-area microplates (Corning) were coated with 2.5 µg/ml of DrSA in 0.1 M sodium carbonate buffer (pH 9.5) and incubated overnight at 4 °C. The plates were blocked with PBS containing 5% skimmed milk for 1.5 h at RT. All plasma samples were analyzed in dilution 1:1,600 in PBS, 1.5 h at RT. Anti-dog peroxidase-conjugated IgG and IgM (abcam, Table S2 ) were diluted 1:50,000 in PBS and incubated 1 h at RT. After every step, plates were washed three times with PBS containing 0.05% Tween-20. TMB substrate solution was added to the wells, developed at RT and stopped after 30 min with 2 M H2SO4. Optical densities were measured at 450 nm in a microplate reader (Synergy HT, BioTek).
10
SARS-CoV-2 RBD Phage ELISA Assay
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A polyclonal phage ELISA was performed using pools of purified phage from each library stock. A 96-Well Half-Area Microplate (Corning, Cat. 3690, New York, NY, USA) was coated overnight at 4 °C, with 30 μL per well of 1 μg/mL SARS-2 RBD (Sino Biological, Cat. 40592-V08H, Beijing, China), and each well was blocked with 5% skimmed milk in PBS (MPBS) for 1 h at room temperature. Phage pools (~1012 phage particles) were also blocked in MPBS for 1 h at room temperature and then blocked phage pools were added to the SARS-2 RBD-coated plate and incubated for 1 h at 37 °C. After washing four times with PBST, the horseradish peroxidase (HRP)-conjugated anti-M13 antibody (1:5000, Sino Biological, Cat. 11973-MM05, Beijing, China) was incubated for 1 h at 37 °C. After washing four times with PBST, a TMB substrate solution (Sigma-Aldrich, Cat. T0440, St. Louis, MO, USA) was added for 8 min, and the reaction was stopped with 1 N sulfuric acid (Merck, Cat. 100731, Darmstadt, Germany). The absorbance was measured at 450 nm using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnydale, CA, USA).
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