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SULT1E1 QC Assay

Activity: [3H]17β-estradiol sulfotransferase (3-sulfate formation)

Incubation conditions
Assay buffer: 0.05 M potassium phosphate pH 7.4, 5 mM MgCl2, 10 mM dithiothreitol
Substrate: [2,4,6,7-3H]17β-estradiol
Incubation volume: 200 µl
Stop reagent: 1 M hydrochloric acid (10 µl)

Important notes:
1. Control cytosol is added to all incubations containing SULT1E1 to bring the total protein concentration (SULT1E1 protein plus control protein) up to 0.5 µg/ml. This is necessary in order to stabilise the SULT1E1 and achieve maximal activity.
2. Using [3H]estradiol from certain suppliers can result in reduced or undetectable activity.

HPLC conditions
Column: HyperClone ODS (5 µm) 250 x 4 mm
Temperature: 40°C
Mobile phase: (isocratic)
49% Water
45% Acetonitrile
6% 50 mM ammonium acetate/50 mM tetrabutylammonium (pH 3.6)
Flow rate: 1.0 ml/min
Run time: 7.0 min
Injection volume: 100 µl
Detection: radiomatic, 3H (liquid scintillation)

Retention times:
17β-estradiol 3-sulfate 4.0 min
17β-estradiol 5.7 min

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