反应详情

Prostaglandin E synthase microsomal fractions are prepared from CHO-K1 cells transiently transfected with plasmid encoding the human mPGES-1 cDNA. Microsomes are then prepared and the PGES assay begins with the incubation of 5 μg/ml microsomal PGES-1 with compound or DMSO (final 1%) for 20-30 minutes at room temperature. The enzyme reactions are performed in 200 mM KPi pH 7.0, 2 mM EDTA and 2.5 mM GSH-reduced form. The enzymatic reaction is then initiated by the addition of 1 μM final PGH2 substrate prepared in isopropanol (3.5% final in assay well) and incubated at room temperature for 30 seconds. The reaction is terminated by the addition of SnCl2 in 1N HCl (1 mg/ml final). Measurement of PGE2 production in the enzyme reaction aliquots is done by EIA using a standard commercially available kit (Cat #: 901-001 from Assay Designs).

1. customMicrosomes are then prepared
2. customfor 20-30 minutes
3. customat room temperature
4. customThe enzymatic reaction
5. customThe reaction is terminated by the addition of SnCl2 in 1N HCl (1 mg/ml final)
6. customMeasurement of PGE2 production in the enzyme reaction aliquots