Practice question
Question
Which enzyme substrate combination gives a yellow product in alkaline pH?
Explanation
Enzyme substrate selection critically determines ELISA readout sensitivity and wavelength. Horseradish peroxidase oxidizes TMB to blue product that turns yellow after acid stop, oxidizes OPD to orange, or oxidizes luminol for chemiluminescence. Alkaline phosphatase hydrolyzes p nitrophenyl phosphate substrate pNPP at alkaline pH 9 to 10 producing p nitrophenol, a soluble yellow product measurable at 405 nanometers. BCIP NBT combination yields insoluble purple precipitate useful for immunoblots and immunohistochemistry. Therefore yellow soluble signal under strongly alkaline conditions characteristically identifies ALP plus pNPP reaction in biochemical assays.
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