| Assay Method Information | |
| | GCPII Activity Assay |
| Description: | Inhibition potencies against GCPII (IC50 values) were determined using previously described methods with minor modification. Rojas et al., 2002. Briefly, reactions were carried out in the presence of NAA-[3H]-G and human recombinant GCPII enzyme in Tris-HCl and CoCl2 at 37° C. for 20 minutes. Reactions were stopped with ice-cold sodium phosphate buffer containing 1 mM EDTA. Aliquots were then transferred to 96-well spin columns containing AG1X8 ion-exchange resin and centrifuged. NAA-[3H]-G was bound to the resin and [3H]-G eluted in the flow through. Columns were washed with formate to ensure complete elution of [3H]-G. The flow-through and the washes were collected, aliquots transferred and dried to completion in a solid scintillator-coated 96-well plate. The radioactivity corresponding to [3H]-G was determined with a scintillation counter. Subsequently, IC50 curves were generated from CPM results. |
| Affinity data for this assay | |
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