| Assay Method Information | |
| | LPD Assay |
| Description: | LPD Assay. Lpd activity was measured by the DTNB assay according to previously published procedures. For preincubation of Lpd with the inhibitor, Lpd was dispensed into the wells, compounds added at specified concentrations, mixed and preincubated at RT for 30 min without any other components. Reaction was started by adding assay mix containing the substrates (lipoamide, NADH) and DTNB and the TNB production was recorded over time at RT in the SpectraMax plate reader at 412 nm. For time-dependent measurements compounds were added to wells containing assay mix and the reaction was started by the addition of Lpd protein and TNB production was followed over time in SpectraMax plate reader at 412 nm. Final concentrations of components in the reaction mixture were as following: 150 μM NADH, 150 μM DTNB, 40 μM NAD+, 75 μM lipoamide in 25 mM potassium phosphate, 1 mM ETDA pH 7.0. Lpd protein was tested at variable concentrations specified in the graphs. Protein concentration was determined by Bradford, Lpd concentration is provided for monomeric enzyme; active species is a dimer and binds 2 molecules of compound per dimer. |
| Affinity data for this assay | |
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