| Assay Method Information | |
| | FABP3, FABP5, and FABP7 Binding Assay |
| Description: | Purified FABPs (3 μM) were incubated with fluorescent probe (500 nM) in 30 mM Tris, 100 mM NaCl buffer (pH 7.5). Compounds to be tested were then added to the wells (0.01-50 μM) and the system was allowed to reach equilibrium by incubating in the dark at room temperature for 20 minutes. Each independent assay included wells containing a strong competitive ligand (arachidonic acid, 10 μM) as a positive control for probe displacement. Loss of fluorescence intensity was monitored with a F5 Filtermax Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using excitation and emission wavelengths appropriate for each respective probe (NBD-stearate ex./em.=465/535 nm, DAUDA ex./em.=345/535 nm, ANS ex./em.=370/465 nm). Following background subtraction, the fluorescence intensity values were normalized and fit to a one-site binding analysis using the GraphPad Prism software (Prism version 7.0 for Mac OS, Graphpad Software Inc., La Jolla, CA, USA) to determine the K, of the tested compounds from the equation K, =IC50/(1+([Probe]/Kd). |
| Affinity data for this assay | |
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