4-Nitrophenyl phosphate → 4-Nitrophenol

Participants

Reaction Steps

To develop color in the two detection systems, coverslips with bound conjugates were rinsed briefly in alkaline buffer (Tris-HCl 100 mM; 100 mM NaLl, 50 mM MgCl2, pH 9.5) and the phosphatase reaction was developed in this buffer containing 40 mM of nitroblue tetrazolium and 40 mM 5-bromo-4-chloro-3 indolyl phosphate (alternatively, it may be possible to utilize the chromogenic phosphatase substrate p-nitrophenyl phosphate which can be enzymatically cleaved by alkaline phosphatase to yield soluble colored p-nitrophenol). Slides were monitored microscopically for the deposition of a purple reaction product at incorporation sites and the reaction was stopped prior to overdevelopment (5-60 minutes) by immersion in stop buffer (10 mM Tris-HCl, 5 mM EDTA; pH 8.0). Coverslips were air dried and mounted onto slides with Permount for evaluation and photomicroscopy.

Provenance & Evidence