| Assay Method Information | |
| | hCTPS1 Biochemical Assay |
| Description: | Purified human CTPS1 protein was prepared in 1× assay buffer to the final working protein concentration required for the reaction. A 2.5 uL volume per well of human CTPS1 protein was mixed with 0.1 uL per well of test compound dissolved in DMSO and pre-incubated at 25 degrees C. for 10 minutes. 2.5 uL per well of the reaction precursors ATP (UltraPure ATP from ADP-Glo™ kit) and UTP were then added and pre-incubated for an additional 10 minutes at 25 degrees C. Finally, the reaction was initiated by the addition of 5 uL of the reaction precursors L-glutamine and GTP. The final concentration of all reaction components in the assay: ATP (120 uM), UTP (160 uM), GTP (60 uM), L-Glutamine (100 uM), DMSO (1%), hCTPS1 (25 nM). This mixture was incubated for an appropriate amount of time within the determined linear phase of the reaction at 25 degrees C. under sealed plate conditions with constant agitation at 500 revolutions per minute (rpm). ADP-Glo™ reagent was added for 60 minutes (10 uL per well) and subsequently ADP-Glo™ development reagent was added for 60 minutes (20 uL per well) prior to signal detection in a microplate reader (Envision Multilabel Reader, Perkin Elmer). Following each reagent addition over the course of the assay, assay plates were pulse centrifuged for 1 minute at 1000 rpm. |
| Affinity data for this assay | |
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