Cypex CYP1B1 QC Assays

Activity: 17b-estradiol 4-hydroxylase

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: none

HPLC conditions
Column: Hypersil BDS-C18 (3 µm) 100 x 4 mm
Temperature: 40°C
Mobile phase: 20 mM potassium phosphate pH 2/methanol/acetonitrile; 48/42/10 (v/v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 8.2 min
Injection volume: 100 µl
Detection: fluorescence l(ex) = 212 nm, l(em) = 323 nm
Retention times:
       4-hydroxyestradiol          3.6 min
     2-hydroxyestradiol          4.3 min
     17b-estradiol                  6.6 min

Effect of phosphate concentration on 17b-estradiol turnover by CYP1B1*3LR Bactosomes
17b-estradiol turnover by CYP1B1 is sensitive to the concentration of phosphate in the incubation.

This assay was carried out at 5 µM 17b-estradiol at a P450 concentration of 5 pmol per 200 µl.

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.

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

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.