4-Nitrophenyl phosphate → 4-Nitrophenol
Reaktionsgleichung
COC1C=C(C2C=CC(N3[N+](C4C=CC([N+]([O-])=O)=CC=4)=NC(C4C=CC=CC=4)=N3)=C(OC)C=2)C=CC=1N1[N+](C2C=CC([N+]([O-])=O)=CC=2)=NC(C2C=CC=CC=2)=N1.P([O-])([O-])(OC1C2C(=CC=C(Br)C=2Cl)NC=1)=O.P([O-])([O-])([O:75][C:76]1[CH:81]=[CH:80][C:79]([N+:82]([O-:84])=[O:83])=[CH:78][CH:77]=1)=O>>[CH:78]1[C:79]([N+:82]([O-:84])=[O:83])=[CH:80][CH:81]=[C:76]([OH:75])[CH:77]=1Komponenten
4
Versuchsvorschrift
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.