Anti-Ro60_316-335 autoantibodies in murine sera were detected by ELISA as described previously (11 (link)). In brief, SSA peptides were absorbed onto Costar EIA/RIA Plates (Corning Icorporated, corning, NY, USA), washed and blocked with 3% BSA in PBS supplemented with 0.05% Tween-20 (PBS-T), incubated with the respective mouse sera (1:200 dilution), and further washed with PBS-T. Bound antibodies were detected by using peroxidase conjugated goat anti-mouse IgG antibodies (Sigma, USA) and tetramethylbenzidine (Solarbio, Beijing, China) as substrate.
Tetramethylbenzidine
Manufactured by Solarbio
Sourced in China, United States
Tetramethylbenzidine is a chemical compound commonly used as a chromogenic substrate in various analytical and diagnostic applications, particularly in enzyme-linked immunosorbent assays (ELISA) and other colorimetric detection methods. It is a sensitive and versatile substrate that undergoes a color change when oxidized, allowing for the detection and quantification of enzymatic activity.
Automatically generated - may contain errors
Lab products found in correlation
Costar eia ria plates, by Corning (2 mentions)
Peroxidase conjugated goat anti mouse igg antibody, by Merck Group (2 mentions)
Methyl blue aqueous solution, by Solarbio (2 mentions)
Glutathione (gsh), by Solarbio (2 mentions)
Bovine serum albumin (bsa), by Solarbio (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Ab97235, by Abcam (1 mentions)
Sulfuric acid, by Solarbio (1 mentions)
Hrp conjugated goat anti mouse igg 31 430, by Thermo Fisher Scientific (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Hrp conjugated anti m13 antibody, by Sino Biological (1 mentions)
Elx800, by Agilent Technologies (1 mentions)
Elisa kit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti mouse igg hrp antibody, by Abcam (1 mentions)
Rbd protein, by GenScript (1 mentions)
Elisa plate, by Corning (1 mentions)
Ammonium acetate, by Thermo Fisher Scientific (1 mentions)
Iodine, by Sinopharm (1 mentions)
Glucose standard, by Merck Group (1 mentions)
11 protocols using tetramethylbenzidine
1
Anti-Ro60 Autoantibody Detection in Mice
2
Serum IgG and Mucosal sIgA Quantification
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Serum IgG and mucosal secretory IgA (sIgA) concentrations were measured using ELISA. Before testing, the samples were briefly diluted with PBS. Microplates containing 100 µL of diluted samples and 5 µg/well of H. pylori lysates were coated overnight at 4 °C. HRP-conjugated goat anti-mouse IgG (31,430, Thermo Fisher) and sIgA (ab97235, Abcam) were used after the plate had been cleaned with PBST. Tetramethylbenzidine (Solarbio, China) was used to view the plates after washing for 15 min in complete darkness. Finally, a solution of 2 M sulfuric acid (Solarbio, China) was used to terminate the process. A microplate reader was used to measure the absorbance at 450 nm.
3
Nanomaterial Synthesis Protocol
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Urea, hydrogen peroxide solution, ammonia solution, tetraethyl orthosilicate, ethanol, sodium hydroxide, nickel sulfate hexahydrate, and iron sulfate heptahydrate were procured from Aladdin Co., Ltd. (Shanghai, China). Tetramethylbenzidine, methyl blue aqueous solution, and glutathione were bought from Solarbio Co., Ltd. (Beijing, China).
4
Nanomaterial Synthesis Protocol
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Urea, hydrogen peroxide solution, ammonia solution, tetraethyl orthosilicate, ethanol, sodium hydroxide, nickel sulfate hexahydrate, and iron sulfate heptahydrate were procured from Aladdin Co., Ltd. (Shanghai, China). Tetramethylbenzidine, methyl blue aqueous solution, and glutathione were bought from Solarbio Co., Ltd. (Beijing, China).
5
Quantitative Membrane Localization Assay
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To quantitatively detect the membrane localization
of mutants and exclude endogenous URAT1, Flag-URAT1-pECFP plasmids
were used in combination with ELISA-based methods. The procedures
were as follows: HEK293 cells were transfected with 500 ng/well N-terminal
Flag-tagged URAT1 or empty vector in 24-well plates. Twenty-four hours
after transfection, the cells were washed with TBS solution and fixed
with paraformaldehyde for 15 min. Then, the cells were blocked with
1% bovine serum albumin (BSA) for 1 h. After that, the cells were
incubated with anti-Flag monoclonal antibody (1:2000, Solarbio, Beijing,
China) for 2 h at room temperature followed by three washes with TBS
solution. Next, the cells were incubated in TBS solution containing
BSA (1%) and HRP-anti-rabbit IgG (1:5000) at room temperature for
1 h and then washed with TBS solution three times. Finally, 150 μL
of the HRP substrate tetramethylbenzidine (Solarbio, Beijing, China)
was added and incubated with the cells for 20 min at room temperature,
and 150 μL of 1 M H2SO4 was added to stop
the reaction. The absorbance was determined at 450 nM using a microplate
reader.
of mutants and exclude endogenous URAT1, Flag-URAT1-pECFP plasmids
were used in combination with ELISA-based methods. The procedures
were as follows: HEK293 cells were transfected with 500 ng/well N-terminal
Flag-tagged URAT1 or empty vector in 24-well plates. Twenty-four hours
after transfection, the cells were washed with TBS solution and fixed
with paraformaldehyde for 15 min. Then, the cells were blocked with
1% bovine serum albumin (BSA) for 1 h. After that, the cells were
incubated with anti-Flag monoclonal antibody (1:2000, Solarbio, Beijing,
China) for 2 h at room temperature followed by three washes with TBS
solution. Next, the cells were incubated in TBS solution containing
BSA (1%) and HRP-anti-rabbit IgG (1:5000) at room temperature for
1 h and then washed with TBS solution three times. Finally, 150 μL
of the HRP substrate tetramethylbenzidine (Solarbio, Beijing, China)
was added and incubated with the cells for 20 min at room temperature,
and 150 μL of 1 M H2SO4 was added to stop
the reaction. The absorbance was determined at 450 nM using a microplate
reader.
6
Rapid Salmonella Detection via Magnetic Separation
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The target bacteria were first specifically separated from the background using immunomagnetic separation (45 min), and the magnetic HRP-bacteria were then separated from the unbound MNPs using viscoelastic particle separation (10 min) and finally detected using enzymatic catalysis colorimetry (5 min). First, 10 µg of the immune HRP-MNPs were incubated with 500 µL of the sample containing different concentrations (101–106 CFU/mL) of Salmonella typhimurium at 15 rpm for 45 min to form the magnetic HRP-bacteria. After that, the mixture of the magnetic HRP-bacteria and the unbound MNPs was resuspended in the viscoelastic solution with 0.5% Tween-20. Then, the magnetic HRP-bacteria were separated from the unbound MNPs using the microfluidic chip. After magnetic separation to remove the viscoelastic solution, the magnetic HRP-bacteria were resuspended in 100 µL of PBS and 50 µL of the suspension was pipetted into the microplate. 100 µL of tetramethyl benzidine from Solarbio was added into the microplate and incubated for 5 min, followed by adding 100 µL of 1 M H2SO4 to terminate the catalytic reaction. Finally, the catalysate was measured using Infinite M200 PRO (Tecan, Männedorf, Switzerland) and the absorbance at the characteristic wavelength of 450 nm was used to determine the concentration of the bacteria.
7
Phage Display Screening for SKOV3 Targets
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After the fourth round of biopanning, 54 phage clones were randomly picked out from titered phage plaques for ELISA. SKOV3 and HEK293 cells were seeded into 96-well plates (4 × 104 cells/well) overnight and then fixed with 4% paraformaldehyde for 20 min at room temperature. 3% H2O2 (100 μl/well) was added, and the plates were placed at room temperature for 30 min to inhibit the activity of endogenous peroxidase. Then cells were blocked with blocking buffer (TBST containing 3% BSA, 250 μl/well) at 37 °C for 2 h. The selected phages (1 × 1010 pfu/well) were added to SKOV3 and HEK293 cells and incubated at 37 °C for 1.5 h. After that, the cells were washed three times with TBST and cultured with HRP-conjugated anti-M13 antibody (diluted at 1:10,000 in 3% BSA, Sino Biological, Beijing, China) for 1 h. Tetramethylbenzidine (100 μl/well, Solarbio Technology Co., Ltd.) were added to the cells and incubated at 37 °C in the dark for 20 min. The incubation was stopped by adding 100 μl/well stop solution. Finally, the 96-well plates were measured at 450 nm using an ELISA reader (Bio-Tek ELX800, USA). Irrelevant phage clone (IRP, an amplified phage randomly selected from the original phage peptide library) and PBS were used as control groups. The relative binding abilities were calculated by ODSKOV3/ODHEK293, the ratio of absorbance over 2.1 was identified as positive clone.
8
Measuring Autoantibodies and Cytokines in Mice
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An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against mRo60_316-335 peptides. The SSA peptides (10 mg/ml in 0.5 M Na2CO3 buffer; pH 9.6) were absorbed onto Costar EIA/RIA Plates (Corning Incorporated, Corning, NY, USA), washed and blocked with 3% BSA in PBS with 0.05% Tween-20 (PBS-T), incubated with the respective mouse sera (1:200), and further washed with PBS-T. Bound antibodies were detected using peroxidase conjugated goat anti-mouse IgG antibodies (Sigma, USA) and tetramethylbenzidine (Solarbio, Beijing, China). Concentrations of IFN-γ, IL-17A, IL-4, and IL-10 in mouse sera were determined by commercially available ELISA kits (Peprotech, USA) according to the manufacturer’s protocols. The detection range of the assay for IFN-γ, IL-17A, IL-4, and IL-10 was 7.5–1,000 pg/ml, 7.5–1,000 pg/ml, 40–5,000 pg/ml, and 15–2,000 pg/ml, respectively. All sera were diluted 1:3 before quantification.
9
ELISA Protocol for SARS-CoV-2 RBD Antibody Detection
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In short, microwells of ELISA plates (Corning, NY, USA) were coated with 50 ng RBD protein each (Genscript, Nanjing, China) at 4 °C overnight. Plates were blocked with 10% FBS in PBS for 1 h at 37 °C, and then incubated with 2-fold serially diluted serum samples for another hour. After washing three times, plates were incubated with rabbit anti-mouse IgG-HRP antibody at a dilution of 1:20,000 (Abcam, Cambridge, UK) at 37 °C for 1 h. Tetramethylbenzidine (Solarbio, Beijing, China) and hydrogen peroxide were used for color development, and the reaction was stopped with 2 M H2SO4. The absorbance was measured at 450 nm, and an optical density at 450 nm (OD450) value greater than 2.1-fold of the background value was regarded as positive [25 (link)].
10
Mycelium Biochemical Characterization
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Mycelium of SJ1 (CGMCC No. 10114) was provided by Shandong Pengbo Biotechnology Co., Ltd. A bicinchoninic acid protein quantification kit, an Ezup column fungal genomic DNA extraction kit, blood vessel collection equipment, a goat anti-rabbit horseradish peroxidase marker, tetramethylbenzidine, 10 mM phosphate buffer solution (PBS) at pH 7.2–7.4, 0.05% tween-20 in PBS and bovine serum albumin were purchased from Beijing Solarbio Science and Technology Co., Ltd., China. Freund’s complete adjuvant, Freund’s incomplete adjuvant, glucose standard (purity ≥ 99%), cholesterol standard (C27H46O, purity ≥ 99%), and L -glutamate standard (purity ≥ 99%) were purchased from Sigma-Aldrich Co. Ltd., United States. Methanol, acetic acid, acetonitrile and ammonium acetate were high-performance liquid chromatography (HPLC) grade and purchased from Thermo Fisher Scientific, China. Industrial alcohol (purity ≥ 95%) were purchased from Jinan Hongzheng Chemical Co., Ltd, China. Hydrochloric acid, sulfuric acid, phenol, petroleum ether, phosphoric acid, potassium iodide, iodine, ammonium ferric sulfate, potassium hydroxide, sodium hydroxide, and all other chemicals and reagents were analytical grade and purchased from Sinopharm, China.
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