| Assay Method Information | |
| | PEPD Enzyme Assay |
| Description: | For the PEPD assay, a solution of substrate (Ala-Pro) was prepared in DMSO. 24 μL of 50 nM recombinant human PEPD were added to a 384-well, black, clear-bottom plate (Corning) with 1 μL dipeptide Ala-Pro (final conc. 40 μM). Alanine liberated was measured as increasing fluorescence signal (Resorufin, Ex/Em: 535/587 nm) recorded at 25° C. using an L-alanine assay kit (Abcam, ab83394) at 25° C. according to manufacturer's instructions. For the AMC reporter assays, experiments were performed with cell lysates. 5 μL solution of substrate (2.5 mM Ala-AMC) was added the mixture of 10 μL HEK293T cell lysates (2.5 mg/ml) and 10 μL of indicated compounds in a 384-well, black, clear-bottom plate (Corning) to initiate the reaction. Substrate cleavage was measured as increasing fluorescence signal (Ex/Em: 380/460 nm) recorded at 25° C. for 20 mins. |
| Affinity data for this assay | |
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