High-binding 96-well ELISA plates (Corning) were coated with 50 μL GP antigens in phosphate-buffered saline (PBS) at 4 μg/mL, and allowed to bind for 1 h at room temperature. After washing, the wells were blocked with PBS containing 3% bovine serum albumin (PBSA) for 1 h at room temperature, followed by washing then incubation with ADI-15946 or one of its mutant derivatives in serial dilutions of PBS. A horseradish-peroxidase conjugated anti-human secondary antibody (Santa Cruz Biotechnology) was added and allowed to bind for 1 h at room temperature and then subsequently detected by ultra-TMB (3,3′,5,5′-tetramethylbenzidine) substrate (ThermoFisher Scientific). Optical density was measured at 450 nm, and absorbance readings were subjected to a nonlinear regression analysis (GraphPad Prism software) to generate binding curves and calculate an EC50 value. ELISA assays were performed in triplicate, across seven 5-fold dilutions, beginning at 1000 ng/ml.
Ultra tmb 3 3 5 5 tetramethylbenzidine substrate
Manufactured by Thermo Fisher Scientific
Ultra-TMB is a substrate solution used for the colorimetric detection of peroxidase enzyme activity in various immunoassays, such as enzyme-linked immunosorbent assays (ELISAs). It provides a sensitive and stable color development reaction for quantitative analysis.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using ultra tmb 3 3 5 5 tetramethylbenzidine substrate
1
ELISA-Based Antibody Binding Analysis
2
ELISA-Based Antibody Binding Analysis
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High-binding 96-well ELISA plates (Corning) were coated with 50 μL GP antigens in phosphate-buffered saline (PBS) at 4 μg/mL, and allowed to bind for 1 h at room temperature. After washing, the wells were blocked with PBS containing 3% bovine serum albumin (PBSA) for 1 h at room temperature, followed by washing then incubation with ADI-15946 or one of its mutant derivatives in serial dilutions of PBS. A horseradish-peroxidase conjugated anti-human secondary antibody (Santa Cruz Biotechnology) was added and allowed to bind for 1 h at room temperature and then subsequently detected by ultra-TMB (3,3′,5,5′-tetramethylbenzidine) substrate (ThermoFisher Scientific). Optical density was measured at 450 nm, and absorbance readings were subjected to a nonlinear regression analysis (GraphPad Prism software) to generate binding curves and calculate an EC50 value. ELISA assays were performed in triplicate, across seven 5-fold dilutions, beginning at 1000 ng/ml.
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