| Assay Method Information | |
| | CYP17-Lyase Assay |
| Description: | Assay method was adopted from a published protocol with some modifications to suit our requirements (Dmitry N Grigoryev et al, Analytical Biochemistry, (1999) 267, 319-330). The conversion of 17-α-hydroxy pregnenolone to Dehydroepiandrosterone is accompanied by the release of acetic acid. In the Cyp17 lyase assay, 17-α-hydroxy pregnenolone labeled with tritium (3H) at position 21 is used as the substrate. Chloroform extraction removes the radioactive steroids and acetic acid is taken into aqueous layer. The tritiated acetic acid released in the assay thus extracted is quantified to determine the enzyme activity. Initial buffer conditions were, 50 mM Phosphate buffer, pH 7.5 was used as the starting buffer for Cyp17 lyase activity based on the data published in US patent publication No. US2004/0198773 A1. This buffer was found to be suitable for regular Cyp17 lyase assay. Human Cyp 17 gene was cloned and expressed in Adenoviral expression system in A549 cell lines. The purified cell membrane preparations were used as the source for Human CYP17 enzyme. Total protein concentration: 8 mg/mL. To identify the appropriate concentration of the enzyme required for the assay, concentration dependent enzyme activity was determined at a substrate (17-α-hydroxypregnenolone[21-3H]) concentration of 0.5 μM (Vincent C. O. Nijar, et al., J Med Chem, (1998) 41, 902-912). The protein activity was found to be in the linear range up to 20 μg, the highest concentration tested. Based on the enzyme concentration curve and stock concentration, 15 μg was selected for the assay. |
| Affinity data for this assay | |
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