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4 was added to stop the reaction. Quantification of colour development and thus enzyme activity was achieved by the measurement of absorbance at 405nm on a Molecular Devices ThermoMax microplate reader.
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48849108969
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note
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Cellular Tie-2 autophosphorylation assay. Tie-2 cell potency was determined using CHOK1 cells stably transfected with human Tie-2. Cells were seeded at 6 × 104 cells/well in 250 μl DMEM, G418, 10% FCS into 96-well plates and grown for 3 days prior to assaying. On the day of assay media was removed and cells were dosed with compound in DMEM plus 1% FCS for 45 min at 37 °C. Cells were washed and lysed in 100 μl lysis buffer (20 mM Tris, pH 7.6, 150 mM NaCl, 50 mM NaF, 0.1% SDS, 1% NP40, 0.5% DOC, 1 mM orthovanadate, 1 mM EDTA, 1 mM PMSF, 30 ml/ml Aprotinin, 10 mg/ml Pepstatin, 10 mg/ml Leupeptin) on ice for 5 min. Lysates were transferred to Quantikine Immunoassay kit for human Tie-2, (R&D systems), and shaken for 2 h at room temperature. Unbound cell lysate was removed by washing four times with the supplied wash buffer, prior to addition of anti-phosphorlyated Tie-2 antibody (4221B Cell Signalling Technologies). Plates were placed on a shaker for 2 h at room temperature. Unbound secondary antibody was removed by washing four times with the supplied wash buffer. P Tie-2 antibody was detected using goat anti-rabbit HRP conjugated antibody (P0448 Dako). Following 2 h of incubation at RT, plates were washed a further four times and the degree of Tie-2 phosphorylation was determined by addition of the colour reagent supplied by R&D Systems. The reaction was stopped after 30 min by addition of stop reagent and optical density was read at 450 nm.
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