Human UGT1A3

UGT1A3 QC Assays

Substrate: 17β-estradiol

Incubation conditions
Assay buffer: 0.1 M Tris-HCl pH 7.5 (at 37°C), 5 mM MgCl₂, 10 µg/ml alamethicin (for kinetics only)
Incubation volume: 200 µl (time/protein linearity) or 1000 µl (kinetics)
Stop reagent: 1 M hydrochloric acid (20 µl for time/protein linearity; 100 µl for kinetics)

HPLC conditions
Column: HyperClone BDS-C18 (3 µm) 100 x 4.6 mm
Temperature: 30°C
Mobile phase: Water/acetonitrile/methanol/100 mM potassium phosphate (pH 2), 40/10/40/10 (v/v/v/v)
Separation type: Isocratic
Flow rate: 1.0 ml/min
Run time: 12 min
Injection volume: 20 µl
Detection: fluorescence λ(ex) = 216 nm, λ(em) = 316 nm

Retention times:
17β-estradiol 3-glucuronide 3.2 min
17β-estradiol 17-glucuronide 4.5 min
17β-estradiol 10.1 min

Substrate: 4-methylumbelliferone (4-MU)

Incubation conditions
Assay buffer: 0.1 M Tris-HCl pH 7.5 (at 37°C), 5 mM MgCl₂, 10 µg/ml alamethicin (for kinetics only)
Incubation volume: 200 µl (time/protein linearity) or 1000 µl (kinetics)
Stop reagent: 1 M hydrochloric acid (20 µl for time/protein linearity; 100 µl for kinetics)

HPLC conditions
Column: HyperClone 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: 15.0 min
Injection volume: 10 µl
Detection: fluorescence λ(ex) = 315 nm, λ(em) = 375 nm

Retention times:
4-MU glucuronide 3.8 min
4-methylumbelliferone 8.9 min