| Assay Method Information | |
| | Biochemical Assay |
| Description: | 5 μl WT full length PAICS (final assay concentration, fac, 2.5 nM) in basic buffer, added to black, non-binding, 384-well plates (Corning #3575), columns 1 to 22. 5 μl basic buffer was added to columns 23+24 (negative control). PAICS stock at 31.9 μM from the PI's lab (Steve Firestine). Basic buffer contained 50 mM Tris-HCl pH8 and 0.5 mM EDTA, fac.1 μl compound in 100% DMSO added per well, or 1 μl 100% DMSO to positive controls (columns 1+2) and negative controls (columns 23+24). Final assay top concentration of compound either 1 mM or 30-μM, serially diluted with half-log dilutions across the plate in duplicate (one 10-point concentration response curve across wells 3 to 12, another across 13 to 22). Compounds pre-incubated with PAICS enzyme for 30 mins at RT. 2 μl CAIR added (fac 10 μM) in basic buffer plus 25 mM MgCl2 and 50 mM KHCO3 fac to all wells. CAIR stock 50 mM from Steve Firestine's lab. 1 hr RT incubation for AIR-CAIR equilibration. NB: 50% CAIR is decarboxylated during equilibration therefore 5 μM remains for the synthetase reaction.2 μl ATP/aspartic acid added (fac 30 μM/180 μM) in reaction buffer to all wells. 30 mins RT incubation for appropriate level of ATP turnover. Reaction buffer contained basic buffer plus 10 mM DTT, 0.01% BSA and 0.01% Brij 35, fac.10 μl ADP detection reagent added to all wells (as per instructions, Transcreener ADP2 FI kit, BellBrook Labs #3013-10K). Incubation for 1 h at RT to allow antibody equilibration. Fluorescence intensity determined using a Tecan Safire2 (excitation at 590 nm, emission at 617 nm). |
| Affinity data for this assay | |
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