| Assay Method Information | |
| | Biochemical humanHSD17B11-RapidFire MS/MS Assay |
| Description: | Estradiol (Sigma, Cat #E8875), NAD (Roche, Cat #10621650001) and recombinant hHSD17B11 (U-Protein Express BV, Netherlands) were diluted in assay buffer (100 mM Tris, Sigma, Cat #T2319; sodium chloride, Roth, Cat #3957.2; 0.5 mM EDTA, Invitrogen, Cat #15575020; 0.1% TCEP, Invitrogen, Cat #T2556; 0.05% BSA fraction V (protease and fatty acid free), Serva, Cat #11945; 0,001% Tween20, Serva, Cat #37470). Compounds were serially diluted in DMSO (Sigma, Cat #5879) and spotted on a 384-well Microplate, PP, V-bottom (Greiner, Cat #781280) plate by a Labcyte Echo 55x (1% DMSO in the Assay). First, 6 μL/well of recombinant hHSD17B11 (35 nM final) dilution was added, followed by 15 min incubation at RT. Second, 6 μL/well of diluted Estradiol (30 μM final) and NAD (0.5 mM final) were added, mixed and incubated for 4 h at RT. 1 μL d4-Estrone (50 nM final; Sigma, Cat #489204) followed by 2.4 μL Girard's Reagent P (6.5 mM final; TCl, Cat #G0030) dissolved in 90% (Sigma, Cat #34860) methanol and 10% formic acid (Merck, Cat #33015) were added to derivatize analytes and stop the enzyme reaction. Incubation was for 12-24 h at RT before adding 70 μL dH2O. The analytical sample handling was performed by a rapid-injecting RapidFire autosampler system (Agilent, Waldbronn, Germany) coupled to a triple quadrupole mass spectrometer (Triple Quad 6500, AB Sciex Germany GmbH, Darmstadt, Germany). |
| Affinity data for this assay | |
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