| Assay Method Information | |
| | Determination of 11β-HSD1 Reductase Activity |
| Description: | First, stock solutions of the compounds were prepared in DMSO at a concentration of 10 μM, of which serial dilutions were made in order to determine the efficacy curve.As for the recombinant 11β-HSD1 enzyme (Cayman Chemical, MI, USA; Item No. 10007815), it was prepared in Tris Buffer 20 mM EDTA 5 mM pH 6.0 (Tris buffer) at different dilutions.Initially, the enzyme concentration to be used in the assays in order to evaluate the biological activity of the compounds was determined. The Cortisol Kit product (Cisbio, MA, USA; Cat no. 62CRTPEG) was used for that purpose.The protocol consisted of preparing the reaction buffer, adding 266 nM cortisone and 333 μM NADPH in Tris buffer. Then, this reaction buffer is added to the wells of the HTRF (Homogeneous Time Resolved Fluorescence) reaction plate together with the enzyme in a 4:1 ratio respectively, and incubated for 2 hours at 37° C. In the case of the control wells, two wells with Tris buffer were added, without compounds.After the incubation time, the reagents of the Cortisol kit were added, Cortisol d2 and Cortisol cryptate in sample ratio, Cortisol d2 and cortisol cryptate 2:1:1, in a quantity according to the size of the well as indicated by the supplier (for the negative control of the kit, the cortisol d2 is replaced by the kit's reconstitution buffer). This reaction is incubated for 1 hour at room temperature in order to later read the fluorescence at 665 and 620 nm.Once the enzyme concentration to be used has been determined, the cortisol kit protocol is continued to determine the efficacy curve for the compounds. As previously done, a plate for HTRF was prepared adding to each well of the reaction buffer, the recombinant 11β-HSD1 enzyme and the compounds to be assayed in a 3:1:1 ratio, respectively. The plate was subsequently incubated at 37° C. for 2 hours.After the incubation time, the reagents of the cortisol kit, cortisol d2 and cortisol cryptate, are added, and were incubated again for 1 hour at room temperature. Finally, the fluorescence of each well was determined at 665 and 620 nm. |
| Affinity data for this assay | |
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