Immunoglobulin (IgG) production was evaluated using an in-house sandwich enzyme-linked immunosorbent assay (ELISA). Splenocytes prepared as described above were incubated with extracts (48 h), 96-well plates were centrifuged (300 g; 10 min), supernatants collected by aspiration, and stored at − 20 °C until analysis. ELISA plates were coated overnight (4 °C) with goat anti-mouse IgG capture antibody (Sigma Aldrich) diluted 1:1000 (10 μg/ml; 75 μl/well) in 0.1 M sodium bicarbonate buffer (pH 9.4). Wells were blotted then blocked with 1% non-fat dried milk (Marvel; 2 h; 37 °C). After washing, diluted cell supernatants were added (75 μl; 2 h; 37 °C) to wells and incubated. Wells were washed with ELISA wash solution and 75 μl of goat anti-mouse IgG secondary detector antibody (HRP-conjugated; 1:2000; Sigma-Aldrich) was added (1 h; 37 °C). Plates were again washed and tetramethylbenzidine (TMB) solution (Sigma-Aldrich) was added, after 10 min H2SO4 (2.5 M) was added and absorbance (450 nm) measured.
Tetramethylbenzidine tmb solution
Manufactured by Merck Group
Sourced in Japan, United States
Tetramethylbenzidine (TMB) solution is a chromogenic substrate used in various laboratory techniques, such as enzyme-linked immunosorbent assays (ELISA). It provides a colorimetric detection system that can be used to measure the presence or activity of enzymes. The TMB solution undergoes a color change reaction when it interacts with the target enzyme, allowing for quantitative or qualitative analysis.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase hrp conjugated anti mouse igg, by GE Healthcare (1 mentions)
Nunc maxisorp, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Chloroauric acid, by Sangon (1 mentions)
Cysteamine, by Maclin Biochemical Technology (1 mentions)
Horseradish peroxidase hrp anti his tag antibody, by BioLegend (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Vector Laboratories (1 mentions)
Sulfuric acid, by Merck Group (1 mentions)
Hrp conjugated anti mouse igg, by Merck Group (1 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
6 protocols using tetramethylbenzidine tmb solution
1
Quantifying Mouse Immunoglobulin Production
2
Mouse Serum ELISA for IL-17A Detection
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ELISA was performed as previously described. Briefly, bovine serum albumin (BSA) conjugated IL-17A1 (AF-588-AB1, lot no. 2839-AB1) and IL17A2 (AF-589-AB1, lot no. 2841-AB1) were synthesized at their N-terminus by suberic acid bis (Peptide Institute Inc., Osaka, Japan). The BSA-IL-17A epitope was coated onto ELISA plates (MaxiSorp Nunc, Thermo Fisher Scientific K.K. Tokyo, Japan) at 10 μg/mL in carbonate buffer overnight at 4 °C. After blocking with a 5% skim milk solution in phosphate-buffered saline (PBS), serial dilutions (1:10–1:31,250) of serum samples from the immunized mice were added to the wells and incubated at 4 °C overnight. After washing with PBS (Phosphate Buffered Saline)–0.05% Tween (PBS-T), the plate was incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (GE Healthcare, Tokyo, Japan) for 3 h at room temperature. After washing, the color was developed using 3, 3′, 5, 5′-tetramethylbenzidine (TMB) solution (Sigma-Aldrich, Tokyo, Japan), and the reaction was stopped using 0.5 N sulfuric acid. The absorbance was read using a microplate reader (Bio-Rad Inc., Hercules, CA). The endpoint titer was expressed as the serum dilution that exhibited half-maximal binding.
3
Gold Nanoparticle Synthesis Protocol
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Chloroauric acid was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Cysteamine and sodium borohydride were provided by Macklin Biochemical Co., Ltd. (Shanghai, China). Tetramethyl-benzidine (TMB) solution was obtained from Sigma-Aldrich (St. Louis, MO). Other chemical reagents were of analytical reagent grade. All solutions were prepared using ultrapure water with a resistivity of 18.2 MΩ cm.
4
Quantifying SARS-CoV-2 Spike Protein Binding
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Human ACE2 protein was immobilized onto a microtiter plate at 5 μg/ml (100 μl/well). Each S protein (prototype and V367F) was added as a ligand at different concentrations, ranging from 0.03 μg/ml to 10 μg/ml, and then incubated for 2 h at 37°C to allow receptor-ligand interaction. The ligand-receptor mixture was then washed three times. One hundred microliters of horseradish peroxidase (HRP) anti-His tag antibody (BioLegend, USA) (diluted 1:20,000) was added to each well and allowed to react for 1 h. After three washes, the signal was visualized using 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Sigma-Aldrich, USA), and a microtiter plate reader recorded the optical density at 450 nm (OD450).
5
Protein Expression Quantification Assay
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Microplates were incubated overnight at 4 °C with 100 nM of carbonate and a bicarbonate-mixed buffer (pH 9.6) and washed three times with 0.1% Tween 20 in phosphate-buffered saline (TPBS) to remove the unattached material. To prevent non-specific protein binding, the microplates were incubated with 5% Skim Milk (LPS Solution) in 0.1% TPBS overnight at 4 °C. After washing three times with 0.1% TPBS, 30 μg of the protein sample was added to each well and incubated overnight at 4 °C. The wells were then washed with 0.1% TPBS and incubated overnight at 4 °C with primary antibodies diluted in PBS (Table S3 ). After washing with PBS, the horseradish peroxidase-conjugated secondary antibody (1:1000; Vector Laboratories) was added and incubated at room temperature for 4 h. To confirm its expression, tetramethylbenzidine (TMB) solution (Sigma-Aldrich) was applied to each well and incubated for 15–20 min at room temperature. To stop the reaction, a stop solution consisting of 2 N of sulfuric acid (Sigma-Aldrich) was used. Finally, measurements were made using a microplate reader at 450 nm. Each analysis was performed in triplicate.
6
Epitope Mapping of Anti-ADAM17 Monoclonal Antibody
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Peptides for epitope mapping of the monoclonal antibody (mAb) against peptide No. 9 (VMYPIAVSGDHENNKMFSNCSKQ) were synthesized (Medical & Biological Laboratories, Nagoya, Japan). The sequences are presented in Supplementary Table 2 . The mAb epitopes were examined using the following two methods:
Inhibition of the antibody binding to rADAM17: ELISA plates were coated with rADAM17 and blocked. The mAb was pre-incubated with each epitope-mapping peptide and then applied to ADAM17-coated ELISA plates. After washing, the bound antibody was detected using horseradish peroxidase (HRP)-conjugated anti-mouse IgG, followed by tetramethylbenzidine (TMB) solution (Sigma, St. Louis, MO).
Binding of the antibody to epitope-mapping peptides: Epitope-mapping peptides were immobilized on a high-binding ELISA plate and blocked. The mAb was applied into each well, washed, and the bound antibody was detected as described above.
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