| Assay Method Information | |
| | LC-MS/MS Based In Vitro Assay |
| Description: | Stock solutions of DHEA (5 mM in DMSO) and compound of interest (5 mM in DMSO) were prepared. For each 60 μL reaction, to a 1.5 mL Eppendorf tube was added 1.2 μL of 5 mM DHEA, 1.2 μL 5 mM compound of interest (for 100 μM final compound concentration) or DMSO, 0.9 μL 6.6 mM PAPS, and 26.7 μL 1× assay buffer. SULT2B1b was diluted to 1.5 μg/μL in protein storage buffer then down to 0.8 μg/μL in 1× activity assay buffer. 30 μL of 0.8 μg/μL SULT2B1b was added to each reaction. Reactions were incubated for 2 hours at 37° C. with shaking at 250 rpm. To quench, 30 μL reaction mix was added to 30 μL ice cold 0.5% HCl in MeCN, then centrifuged at 13,000 xg at room temperature for 3 min. 50 μL supernatant was transferred to an empty Eppendorf tube and submitted for LC-MS/MS quantification (UIUC Metabolomics Center), along with 5 mM DHEA sulfate standard. Controls: Reaction mix made with DHEA and DMSO in place of compound of interest (DHEA control). |
| Affinity data for this assay | |
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