Cypex CYP1A2 QC Assays

Activity: 7-ethoxycoumarin O-deethylase

Incubation conditions
Assay buffer: 0.05 M potassium phosphate pH 7.4, 5 mM MgCl₂
Incubation volume: 200 µl
Stop reagent: 1 M hydrochloric acid (20 µl)
Internal standard: 4-methylumbelliferone (10 µl of 0.1 µg/ml)

HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 30°C
Mobile phase: 0.05% orthophosphoric acid/acetonitrile (gradient separation)
Flow rate: 1.0 ml/min
Run time: 14.5 min
Injection volume: 50 µl
Detection: fluorescence l(ex) = 324 nm, l(em) = 458 nm
Retention times:
       umbelliferone                5.2 min
     4-methylumbelliferone    7.0 min
     7-ethoxycoumarin         10.3 min

Effect of phosphate concentration on 7-ethoxycoumarin turnover by CYP1A2R Bactosomes
ECOD activity is relatively insensitive to the concentration of phosphate in the incubation.

This assay was carried out at 20 µM 7-ethoxycoumarin at a P450 concentration of 0.5 pmol per 200 µl.

Activity: Phenacetin O-deethylase

Incubation conditions
Assay buffer: 0.05 M potassium phosphate pH 7.4, 5 mM MgCl₂
Incubation volume: 200 µl
Stop reagent: 1 M hydrochloric acid (20 µl)
Internal standard: 3-acetamidophenol (10 µl of 10 µg/ml)

HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 30°C
Mobile phase: 0.05% orthophosphoric acid/acetonitrile (gradient separation)
Flow rate: 1.0 ml/min
Run time: 18.5 min
Injection volume: 50 µl
Detection: UV, l = 250 nm
Retention times:
       4-acetamidophenol            4.2 min
     3-acetamidophenol            6.1 min
     phenacetin                       12.5 min

Activity: 7-ethoxyresorufin O-deethylase

Incubation conditions
Assay buffer: 0.1 M potassium phosphate pH 7.4, 5 mM MgCl₂
Incubation volume: 200 µl
Stop reagent: methanol (100 µl)
Internal standard: none

HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 35°C
Mobile phase: 20 mM potassium phosphate pH 7/methanol 58/42 (v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 5.5 min
Injection volume: 75 µl
Detection: fluorescence l(ex) = 530 nm, l(em) = 580 nm
Retention times:
     resorufin                         4.0 min

The substrate 7-ethoxyresorufin and the metabolite resorufin are both available from Cypex Ltd.

Details of a plate reader assay for CYP1A2 activity using 7-ethoxyresorufin can be found here.

Other Substrates

Important: By purchasing this product from Cypex you accept the standard terms and conditions of supply, details of which may be provided upon request.

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Made under licence from BTG International Ltd (AU730155, EP0914446, US6566108 and other patents pending).

United States Patent Nos. 5,420,027 or 5,240,831, Canada Patent No. 2100245 and other patents pending.