| Assay Method Information | |
| | Binding assays for FABP3, FABP4, FABP5 and FABP7 |
| Description: | Binding assays for FABP3, FABP4, FABP5 and FABP7 were carried out by fluorescence titrations. His-tagged FABPs were bacterially expressed in E. coli, purified using Ni Sepharose beads, and the equilibrium dissociation constants (Kd) that characterize their interactions with different inhibitor compounds were measured by fluorescence competition assays. The method entails two steps as described in e.g., Lin, Q. et al., “Ligand selectivity of the peroxisome proliferator-activated receptor alpha,” Biochemistry 38, 185-190, doi:10.1021/bi9816094 [pii](1999). In the first step, Kd for the association of the protein with the fluorescent fatty acid probe ANS was measured. Protein (2 μM) was titrated with ANS from a concentrated solution in DMSO. Ligand binding was monitored by following the increase in the fluorescence of the ligand upon binding to the protein, and Kd for the association of ANS with the each FABP was computed from titration curves as described in e.g., Norris, A. W. & Li, E., “Fluorometric titration of the CRABPs,” Methods Mol Biol 89, 123-139 (1998)). In the second step, Kds for binding of non-fluorescent ligands were measured by monitoring their ability to displace ANS in the binding pocket of the protein. |
| Affinity data for this assay | |
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