Activity: 4-nitrobenzaldehyde oxidase
Incubation conditions
Assay buffer: 0.05 M Hepes pH 7.4, 1 mM EDTA, 2 mM DTT, 1 mM NAD+
Incubation volume: 500 µl
Stop reagent: 1 M hydrochloric acid (62.5 µl)
HPLC conditions
Column: Gemini C18 (3 µm) 100 x 4.6 mm
Temperature: 60°C
Mobile phase: 0.5% trifluoroacetic acid/methanol/water 40/46/14 (v/v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 4.0 min
Injection volume: 100 µl
Detection: UV, λ = 264 nm
Retention times:
4-nitrobenzaldehyde 2.7 min
4-nitrobenzoic acid 3.1 min