The iNOS contents were determined using the method described previously by Marshall et al. [29 (link)] and modified by Saia et al. [18 (link)]. The macrophage homogenates were diluted in phosphate-buffered saline (PBS 0.1 M, pH 7.2) and incubated overnight at 4°C in Nunc Maxisorb plates (Life Technologies, Paisley, UK). The wells were washed with PBS containing 0.05% Tween-20 (PBS-T) and blocked with 1% (w/v) bovine serum albumin (BSA) for 1 h at room temperature. Subsequently, the polyclonal rabbit anti-iNOS antibody (1:1000; Sigma) was added for 1 h at room temperature. After washing, the biotinylated anti-rabbit IgG secondary antibody (1:200; Vector Laboratories) was added to each well, incubated with avidin-HRP (1:5000; Sigma) for 30 min, and the colour developed by addition of 3,3′,5,5′-tetramethylbenzidine substrate (TMB; BD Biosciences, Franklin Lakes, NJ, USA). The reaction was stopped with 2 N H2SO4 and the optical density (OD) reading at 450 nm was taken. The sample levels of iNOS were expressed as OD normalized to the respective total protein concentrations.
Tetramethylbenzidine tmb substrate
Manufactured by BD
Sourced in United States
Tetramethylbenzidine (TMB) substrate is a chromogenic substrate commonly used in various immunoassays, such as ELISA, to detect and quantify the presence of specific analytes. It undergoes an enzymatic reaction, resulting in a color change that can be measured spectrophotometrically. The core function of TMB substrate is to provide a sensitive and reliable means of detecting the presence and concentration of target molecules in a sample.
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Lab products found in correlation
Non fat dry milk, by GE Healthcare (2 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (2 mentions)
Avidin hrp, by Merck Group (1 mentions)
Anti inos antibody, by Merck Group (1 mentions)
Biotinylated anti rabbit igg secondary antibody, by Vector Laboratories (1 mentions)
Anti mouse igg whole molecule peroxidase, by Merck Group (1 mentions)
Microplate reader, by Tecan (1 mentions)
Blotting grade blocker, by Bio-Rad (1 mentions)
Anti mouse ige, by Southern Biotech (1 mentions)
Ovalbumin grade 6, by Merck Group (1 mentions)
Anti mouse igg2b, by Southern Biotech (1 mentions)
Anti mouse igg2c, by Southern Biotech (1 mentions)
Goat anti mouse igg, by Southern Biotech (1 mentions)
Maxisorp, by Thermo Fisher Scientific (1 mentions)
Varioskan, by Thermo Fisher Scientific (1 mentions)
Neutravidin hrp, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated streptavidin, by Cytiva (1 mentions)
Elx808 absorbance microplate reader, by Agilent Technologies (1 mentions)
High binding microplate, by Greiner (1 mentions)
Hace2, by R&D Systems (1 mentions)
14 protocols using tetramethylbenzidine tmb substrate
1
Quantitative Measurement of iNOS
2
Murine Serum IgG ELISA
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To determine IgG-specific antibody in murine sera, the enzyme-limited immunosorbent assay (ELISA) method was performed. Briefly, the wells were coated with 250 ng purified RgpA in carbonate-bicarbonate buffer (pH 9.5) for 16 h at 4°C or with 100 μl of formaldehyde-fixed P. gingivalis W83 cells (OD600 = 0.5) for 1 h at 37°C and then for 16 h at 4°C. The next day, wells were washed 3 times with PBS and blocked in 2% BSA in PBS. After several washes, 4-fold dilutions of murine sera were added to the wells and incubated for 1 h in 37°C. Following washing 3 times with PBS, sheep anti-mouse IgG peroxidase-conjugated antibody (Sigma-Aldrich; 1:4,000 diluted in 1% BSA in PBS) was added for 1 h at 37°C. Wells were washed 5 times, and 3,3,5,5-tetramethylbenzidine substrate (TMB) was added (BD Biosciences). The reaction was stopped with 2N sulfuric acid, and the color intensity was recorded at 450 nm.
3
Antigen-specific Antibody Detection in Immunized Mice
4
MERS and HCV Protein Binding Assay
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Example 8
The wells of 96-well plates are coated with MERS spike protein or HCV E1E2 protein. The coated plates are blocked (BioLegend, San Diego, Ca), incubated with serially diluted Receptor-Fc-HS proteins for 2 hr at RT, incubated with 1/1000 dilution of horseradish peroxidase-conjugated goat-anti human Fc (LSBio, Inc) for 1 hr at RT, washed, and then tetramethylbenzidine (TMB) substrate (BD Bioscience) was added. The reaction was stopped with 2N H2SO4, and the absorbency read at 450 nM.
5
Antigen-Specific Antibody Detection Assay
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Nunc Maxisorp 96-well plates were coated with 10 μg/mL Ovalbumin (grade VI, SIGMA-ALDRICH), goat anti- mouse IgG (1 μg/mL) (Southern Biotech) and incubated overnight at 4°C. Plates were washed 3 times with 0.5% Tween-20 in PBS and blocked with 200 μl of 2% blotting-grade blocker (Biorad). Mouse serum samples were serially diluted in blocking buffer and incubated on blocked plates. Antigen-specific serum antibodies were detected using horseradish peroxidase (HRP)-conjugated antibodies (anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c, and anti-mouse IgE) (Southern Biotech) at 1:5000 dilution in blocking buffer. Incubation of serum samples or antibodies was conducted at room temperature. HRP activity was detected using 100 μL of tetramethylbenzidine (TMB) substrate (BD Biosciences) and stopped using 50 μL 2N H2SO4. Developed plates were recorded using BioRad spectrophotometer at 450 nm with correction at 595 nm by subtraction.
6
Oral Gavage for Plasma FSA and HRP
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Mice received, by oral gavage, 5 µL/g of the animal weight of a solution containing 10 mg/mL of fluorescein-5.6 sulfonic acid (FSA, Thermo Fisher Scientific, Illkirch-Graffenstaden, France) and horseradish peroxidase (HRP, Sigma-Aldrich, Saint-Quentin-Fallavier, France) diluted in 0.5% of carboxy-methyl-cellulose. Blood was collected from the tail vein 2 h before and afterthe gavage. Plasma was isolated by centrifugation at 3200 rpm for 10 min andFSA concentration was determined by measuring the fluorescence intensity at 488 nm using a spectrofluorometer (Varioskan, Thermo Fisher Scientific, Illkirch-Graffenstaden, France). HRP activity was determined by enzymatic assay with tetramethylbenzidine (TMB) substrate (BD Biosciences).
7
Quantifying mAb Affinity to H7N9 HA via ELISA
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The approximate affinity of mAb against H7N9 HA was determined using an indirect ELISA method. Generally, 100 ng of purified H7N9 rHA was coated on the bottom of the 96-well plate for 1 h at room temperature. The plate was blocked with 1% BSA in PBS for 1 h at room temperature. Subsequently, a series of two-fold dilutions (2000–12,800×) of mAbs were added to each well of the plate to incubate with H7N9 rHA for 1 h at room temperature. The 96-well plate was washed three times with PBS containing 0.05% Tween 20 (PBST). Next, the horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was added to each well of the plate, followed by incubation at room temperature for 1 h. Finally, 100 μL of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (BD Bioscience, USA) was added to each well of the plate for signal detection. Absorbance at 450 nm was measured and recorded by ELISA reader.
8
Mouse/Human IL1RAPL1 Binding Assay
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Mouse or human IL1RAPL1/Fc chimera (Sino Biological Inc.) were immobilized on 384 maxisorp plates (NUNC) at a concentration of 0.1 mg/well in 25 ml PBS overnight at 4 C. After blocking with 90 ml PBT-buffer (0.5% BSA, 0.1% Tween 20 in PBS) for 1 h and 4 C, a 1:1 dilution series of recombinant mouse or human His-Ub-IL-38-Avi fusion protein (0, 0.031, 0.063, 0.125, 0.25, 0.5 and 1 mM) were added to the wells and incubated for 2 h at 4 C in PBT buffer. After IL-38 binding, wells were washed 4 times with 90 ml PT buffer (0.1% Tween 20 in PBS). Subsequently, 25 mL of High Sensitivity NeutrAvidin-HRP (Thermo Fisher Scientific, diluted 1:2000 in PBT buffer) was added to the wells. After incubation for 1 h at 4 C, the plate was washed 4 times with PT buffer and 25 ml freshly prepared Tetramethylbenzidine (TMB) substrate (BD Biosciences) was added. The reaction was stopped with 25 mL 1.0 M H3PO4. Signals were read spectrophotometrically at 450 nm (Epoch, BioTek). Plotted values were corrected for background binding of mouse His-Ub-IL-38-Avi to BSA coated wells.
9
Quantifying Chicken IFNγ by ELISA
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Supernatants from the stimulated splenocytes were harvested and examined by sandwich ELISA. Briefly, anti-chicken IFNγ (2 μg/ml, Invitrogen) was coated onto 96-well maxisorp ELISA plates (Thermo Fisher Scientific). The coated plates were blocked at room temperature with PBS containing 3% BSA for 1 h. A 1:2 dilution of the supernatants was made in PBS buffer containing 3% BSA. The plates were then incubated with the diluted supernatants for 2 h at room temperature. Detection was carried out using biotinylated anti-chicken IFNγ detection antibody (1 μg/ml, Invitrogen) for 1 h at room temperature followed by HRP conjugated streptavidin (1:1000 dilution, Amersham) for another 1 h at room temperature. A 100 μl of Tetramethylbenzidine (TMB) substrate (BD biosciences) was added for 10 min. The reaction was stopped using 2 M H2SO4 and read at wavelength 450 nm in ELx808 Absorbance Microplate Reader (BioTek).
10
ACE2-RBD Binding Quantification by ELISA
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ACE2 binding to the produced RBD variant antigens was analyzed by ELISA. Briefly, 0.1 ug of RBD per well was immobilized on a high binding Microplate (Greiner Bio-One GmbH, Austria.) and incubated ON at 4 °C. Next, plates were blocked with 5% non-fat milk in PBS 0.05% Tween20 for 2 h at 37 °C and then washed with PBS and 0.05% Tween20. Plates were then incubated with hACE2 (R&D, Minneapolis MN, USA) for 2 h at 37 °C and another washing step was performed. Finally, plates were incubated with anti-hACE2 (R&D Systems, Cat#: AF933. Lot: HOK0620051) diluted to a concentration of 0.5 µg/ml in 1% non-fat milk PBS 0.05% Tween 20 for 2 h at 37 °C. Detection was performed with a secondary Ab conjugated to HRP (Agilent DAKO, Santa Clara, CA, USA, Cat# P0449, Lot:41308941) at a dilution: 1/1000 in 1% non-fat milk PBS 0.05% Tween 20 for 1 h at 37 °C and visualized with tetramethylbenzidine (TMB) substrate (BD biosciences, Franklin Lakes, New Jersey, USA).
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