| Assay Method Information | |
| | In Vitro ADA Activity Assay |
| Description: | For the ADA activity assay, a reaction comprising 50 mM potassium phosphate buffer (pH 7.2), 0.1 mM Ara-A or adenosine, and 20 mg ADA was conducted at 30 °C for 30 min. Reactions were terminated by the addition of an equivalent volume of methanol, followed by centrifugation to get rid of protein. The reactions were analyzed by HPLC (Shimadzu LC-20A), equipped with a reversephase C18 column (Inertsil ODS-3, 4.63250 mm, 5 mm), at the elution gradient of 5%-20% methanol:0.15% trifluoroacetic acid (TFA; HPLC grade) for 10 min, then the ratio of 20% methanol:0.15% TFA was continued until 30 min. The elution was monitored by a DAD at 260 nm with a flow rate of 0.5 mL/min. Assays were measured at ten different concentrations of substrate (adenosine, Ara-A) ranging from 10 to 100 mM in 50 mM potassium phosphate (pH 7.2) at 30 °C, and the assays were repeated at least three times. The inhibitory activity of PTN was assayed by adding differentconcentrations (final concentrations 1, 2, and 5 nM) to the reaction mixture described above. |
| Affinity data for this assay | |
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