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| | Fluorescence Resonance Energy Transfer (FRET) Assay |
| Description: | A total of 30,000 chemical compounds (DiverSet Chemically Diverse Library and Combichem Library, ChemBridge Corp.) were screened for SrtA inhibition using an automated robotic system at the UCLA Molecular Screening Shared Resource facility. A FRET assay was used in high-throughput screening in multi-well plates (384 wells per plate). The assay monitors the SrtA-catalyzed hydrolysis of an internally quenched fluorescent substrate analog. Purified SrtA (>95% homogeneity and proper folding was confirmed by 1D H NMR) was incubated with test compound solution. Then fluorescent substrate solution was added to the mixture to initiate the catalysis. The FRET assays were monitored by a Flex Station II plate reader (Molecular Devices) with an excitation and emission wavelengths of 335 and 420 nm, respectively. The assay mixture was measured again after 5 h for end-point reading. For the top ten lead compounds, IC50 was determined by fitting three independent sets of data to the equation. Some of the inhibitors tightly bind to the enzyme such that their IC50 values are lower than the enzyme concentration used in the assay. To accurately define their potency the IC50 values of these compounds were measured at different enzyme concentrations. |
| Affinity data for this assay | |
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