| Assay Method Information | |
| | In Vitro Phosphatase Assay |
| Description: | Assays with and without inhibitor were performed in Corning 96-well clear bottom plates having a nonbinding surface, with a total assay volume of 200 μL. The assay buffer contained 100 mM Tris, 100 mM NaCl, 1 mM EDTA at pH 7.0 and 2.2 mM pNPP as the substrate. Serial dilutions of each of the RR601 and NSC-87877 stocks were performed such that upon the addition of 4 μL of a respective inhibitor stock to the assay buffer, the resulting range in assay inhibitor concentrations were 0.03 μM to 100 μM for RR601 and 0.1 μM to 300 μM for NSC-87877. The reaction was initiated upon addition of 4 μL of a 5.0 μM PTP1B stock, which was dispensed to each of the wells except the blanks using a single channel pipette. The resulting PTPB1 concentration in the assay buffer was 0.1 μM. The plate was returned to the plate reader, shaken, and absorbance recorded at 25 °C every thirty seconds over a 10 mins period when RR601 (Fig. 8a) was used, or for 60 mins when NSC-87877 was used as the inhibitor. |
| Affinity data for this assay | |
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