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Assay Method Information |
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| | Colorimetric Malachite Green Assay |
| Description: | The compounds were first mixed with tetrasodium PPi (final concentration of 100 μM) at concentrations specified in the text in the reaction buffer (20 mM Tris-HCl pH 7.5 adjusted at room temperature, and 1 mM MgCl2 for MtPPiase, EcPPiase, ScPPiase or 1 mM MnCl2 for SaPPiase). The total reaction volume was 30μL. The compounds were first tested for inhibition at 4 different concentrations (100, 200, 400, and 600 μM). In each case we added 2 μL of different compound stocks or 2 μL of DMSO without compound (for 0 μM compound assays), to ensure the same DMSO concentration in all the reactions, due to a minor inhibitory effect of DMSO. To initiate the reaction, PPiase (at the final concentration of 2 nM for all PPiases used except for SaPPiase which was used at 0.67 μM for comparable activity) was added to the mixture. The reaction was incubated for 8 min at roomtemperature and then quenched by addition of 150 μL of the malachite green reagent. After |
| Affinity data for this assay | |
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