| Assay Method Information | |
| | In Vitro Determination of Human QC Inhibitory Activity |
| Description: | The inhibitors to be tested were dissolved in DMSO, as a 250 mM stock solution and stored at −20° C. until further use. For the assay, compounds were diluted in the provided assay buffer to the desired concentrations, from 100 μM to 0.1 nM, so that in all wells a final DMSO concentration of 0.6% was present. The enzyme solution was prepared immediately before use, by diluting 10 μL of recombinant enzyme (component C) in 3.99 mL of assay buffer (component E) and it was stored on ice. The substrate solution was prepared by diluting 50 μL of glutaminyl cyclase substrate (0.5 mM, component A) in 4.95 mL of assay buffer (component E). The developer solution was prepared by diluting 50 μL of glutaminyl cyclase developer (component D) and 50 μL of 1-benzylimidazole (component F) in 4.9 mL of assay buffer (component E), and was kept on ice until use. In brief, 10 μL of the solution of the inhibitor were placed into wells of a black 96-well plate and combined with 40 μL of the diluted enzyme solution. To start the reaction 50 μL of glutaminyl cyclase substrate solution were added. After 30 min incubation at 37° C., 50 μL of developer solution were added and mixed. After another 30 min incubation at 37° C., the signal was measured at EX485/EM535 at Cytation 5 multimode reader. |
| Affinity data for this assay | |
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