| Assay Method Information | |
| | Enzyme Activity Assay |
| Description: | An enzyme activity assay for BLVRB was conducted. Since BLVRB catalyzes the NAD(P)H dependent readuction of FMN, changes in the NAD(P)H concentration in the presence of FMN can be used to measure enzyme activity in the absence (control) and presence of drug candidates. The IC50 (the concentration of the drug candidate at which BLVRB activity to convert NADPH to NADP+ in the presence of FMN was reduced to 50%) was used as the selection criterion for the screenings of more potent inhibitory molecules with lower IC50 value. Native form of BLVRB (without NADP+) was used for enzyme activity assay. Assay was performed by monitoring the rate of oxidation of NADPH at 340 nm with Gemini EM Microplate Reader. All assays were operated at 25° C. in 100 mM Phosphate-buffered saline, 0.01% of triton X-100, 100 μM FMN, 100 μM NADPH, 1 μM BLVRB and variable concentration of compounds of the present disclosure. The control reactions were carried out in the absence of compounds of the present disclosure to compare the effectiveness of the drug. FMN and NADPH were freshly constructed with 10 mM stock daily, calculated by UV/Vis spectrometer. In each case, FMN was added to initiate the reaction and measured for 30 minutes. The concentration of the compounds of the present disclosure was sequentially decreased in the order of 100 μM, 25 μM, and 5 μM. |
| Affinity data for this assay | |
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