| Assay Method Information | |
| | In Vitro TMA Inhibition |
| Description: | Choline TMA lyase enzymatic activity assays were conducted to measure the conversion of isotope-labeled d9-choline to d9-TMA. Upon thawing an aliquot of frozen clarified lysate on ice and gentle mixing, clarified lysate was diluted to 5 mg/mL in lysis buffer and the necessary co-factors were added (final concentration: 1 mM SAM, 10 mM NaDT, and 2 mM NADH). Potential inhibitor compounds (typically dissolved in H2O) were serially diluted (10-fold) and added to individual reaction mixtures (done in triplicate). The mixture was then allowed to incubate for at least 15 minutes at 22° C. before the addition of substrate (300 μM final d9-choline) to initiate the reaction, which was carried out for 2 hours at room temperature (22° C.) in the dark in gas tight sealed reaction vials. The reactions were quenched with the addition of 200 μL of 1 M NaOH and 0.5 μM of the internal standard ([13C3 15N1]TMA), vortexing and placement in ice. d9-TMA production was quantified using mass spectrometry by measuring the peak area ratio of the precursor to product ion transition of d9-TMA (m/z 69→49) to the internal standard [13C3 15N1]TMA (m/z 64→47). |
| Affinity data for this assay | |
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