To determine the binding avidity of mouse serum samples, serially diluted mouse serum samples were incubated at 37 °C in a 96-well ELISA microplate (Thermo Fisher, USA) coated with 0.1 µg/well SARS-CoV-2 S protein. After 1 h of incubation, the plates were washed three times with wash buffer and then incubated with a 1:4000 dilution of horseradish peroxidase-conjugated goat anti-human IgG (Genscript, China) for 1 h at 37 °C. After five washes, 100 µl of freshly prepared 3,3’,5,5’-tetramethylbenzidine substrate (Wantai, China) was added to each well, followed by incubation for 15 min at room temperature in the dark, and then 50 μl/well of 2 M H2SO4 was added to stop the reaction. The result is presented as absorbance read at 450 nm subtracted from absorbance at 620 nm.
96 well elisa microplate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 96-well ELISA microplates are a laboratory equipment designed for enzyme-linked immunosorbent assay (ELISA) experiments. These microplates provide a standardized format with 96 individual wells, allowing for the simultaneous processing of multiple samples and controls.
Automatically generated - may contain errors
Lab products found in correlation
5 5 tetramethylbenzidine tmb substrate, by Merck Group (2 mentions)
Hrp conjugated goat anti human total ab, by Thermo Fisher Scientific (2 mentions)
Imark microplate reader, by Bio-Rad (1 mentions)
Streptavidin agarose bead suspension, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated streptavidin, by Merck Group (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Bradford protein assay, by Thermo Fisher Scientific (1 mentions)
Softmax pro 6, by Molecular Devices (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
5 protocols using 96 well elisa microplate
1
Quantifying SARS-CoV-2 Antibody Avidity
2
Colorimetric Detection of HRP-Oligonucleotide Conjugates
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HRP-oligonucleotide conjugates for immobilization were prepared as previously described18 (link) using the oligonucleotides shown in Table 1 . The conjugates were immobilized through biotin-streptavidin interactions on the surface of either streptavidin-precoated 96-microwell plates, or the streptavidin agarose bead suspension (both from Thermo Fisher Scientific). HRP was released from the surface attachment as the result of REase cleavage of probes and linkers. Reaction mixtures containing released HRP were transferred to wells of a 96-well ELISA microplate (Thermo Fisher Scientific). Then 100 μL of the BioFX TMB One Component HRP Microwell Substrate (SurModics, Eden Prairie, MN) was added into each well. The HRP-generated signal was quantified after 2–5 min incubation at room temperature by the blue color formation that was measured colorimetrically at the wavelength of 655 nM (OD655), using an iMark Microplate Reader (Bio-Rad).
3
Quantification of TDP-43 and Aβ Interactions
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The concentration of freshly purified TDP-43_FL and truncation proteins were quantified by Bradford protein assay (Thermo Fisher Scientific) and the same mole number of each variant at 1 μM (200 pmole in 200 μL) was immobilized in 96-well ELISA microplates (Thermo Fisher Scientific, USA) by incubation overnight at 4 °C. HFIP and NaOH-treated biotin-labeled Aβ40 was prepared in 10 mM Tris at pH 7.8 to make a series of Aβ solution with concentrations ranging from 0 to 38 μM. After immobilization, the plate was blocked for 2 h at RT with 5% skim milk in TBST (50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 0.1% Tween-20). The plates were washed and probed by biotin-Aβ40 solution for 2 h at RT. After wash, the plates were subjected to HRP-conjugated Streptavidin (1:5,000, Merck Millipore) for 1 h at RT. After another wash, the color was developed by adding 100 μL 3,3, 5,5 -tetramethyl benzidine (Merck Millipore). The reaction was stopped with 100 μL 250 mM HCl and the absorbance was recorded at 450 nm by software SoftMax Pro 6.3 in SpectraMax M5 (Molecular Device). Data were plotted and analyzed in GraphPad Prism 7.0.
4
SARS-CoV-2 Serum Antibody ELISA Assay
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Serum antibodies against SARS-CoV-2 were determined using an indirect ELISA. The 96-well ELISA microplates (Thermo Fisher Scientific Korea, Ltd.) were coated overnight at 4°C with 100 μL per well of 2 μg/mL of plant- and E. coli -expressed rNPs. The microplates were washed three times with washing buffer of PBS-T (0.05% Tween 20) and blocked with blocking buffer (PBS-T containing 5% of skim milk) for 2 h at 37°C. After four washes, the specimens were diluted 100-fold with blocking solution and incubated at 37°C for 2 h. The plates were then washed five times with washing buffer. Following this step, HRP-conjugated goat anti-human total Ab (Thermo Fisher Scientific; catalog no. 31418) was diluted in blocking solution (1:40,000) and added at a 100 μL volume per well and incubated at 37°C for 1 h. After an extensive washing step, 50 μL of 3,3′,5,5′-tetramethylbenzidine substrate (TMB; Sigma-Aldrich, USA) was added to each well at room temperature in the dark. After 30 min, the reaction was stopped with 25 μL of 1 M H2SO4, and the absorbance at 450 nm was measured in each well. The samples were tested in triplicate.
5
SARS-CoV-2 Serum Antibody ELISA Assay
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Serum antibodies against SARS-CoV-2 were determined using an indirect ELISA. The 96-well ELISA microplates (Thermo Fisher Scientific Korea, Ltd.) were coated overnight at 4°C with 100 μL per well of 2 μg/mL of plant- and E. coli -expressed rNPs. The microplates were washed three times with washing buffer of PBS-T (0.05% Tween 20) and blocked with blocking buffer (PBS-T containing 5% of skim milk) for 2 h at 37°C. After four washes, the specimens were diluted 100-fold with blocking solution and incubated at 37°C for 2 h. The plates were then washed five times with washing buffer. Following this step, HRP-conjugated goat anti-human total Ab (Thermo Fisher Scientific; catalog no. 31418) was diluted in blocking solution (1:40,000) and added at a 100 μL volume per well and incubated at 37°C for 1 h. After an extensive washing step, 50 μL of 3,3′,5,5′-tetramethylbenzidine substrate (TMB; Sigma-Aldrich, USA) was added to each well at room temperature in the dark. After 30 min, the reaction was stopped with 25 μL of 1 M H2SO4, and the absorbance at 450 nm was measured in each well. The samples were tested in triplicate.
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