| Assay Method Information | |
| | pH-Stat Assay |
| Description: | A NaOH stock solution (50 mM) was standardized with KHP then diluted in Millipore water (10-fold serial dilutions) then used to hold the pH constant (at 8.0) in fixed concentrations of 5 mM for diC4PC, 150 mM NaCl, and 5 mM CaCl2 (Ca2+ for PLD_SC only). The diC4PA product decreased the pH and was in a 1:1 ratio with moles of NaOH needed to stabilize the pH at 8.0. This pH is appropriate as the pKa2 of the diC4PA side chain is ~6.8 so that all of this product will be titrated at pH 8.0 (14). Once the baseline-specific activity was determined for the enzyme, known amounts of inhibitors ranging from 0.1 nM to 0.1 mM concentration were added. Initial molecule screening was carried out with small molecule inhibitors listed in Table 1, in the micromolar range (~10 data points in duplicate). Then, full duplicate curves were generated higher or lower than these initial concentrations depending on the efficiency of each individual compound. In these assays, between 1.5 and 2.5 µg of PLD enzyme |
| Affinity data for this assay | |
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