| Assay Method Information | |
| | Ligand Binding Assay |
| Description: | In brief, both wild-type and mutant hFABP5 were expressed and purified to homogeneity as described above and dialyzed in PBS (pH 8.2). Binding affinity(KD) was derived by monitoring maximal fluorescence intensity of a constant concentration of 500 nM 1,8-ANS with increasing protein concentrations ranging from 20 nM to 424 μM. Blank measurements obtained from protein-only samples were subtracted at each protein concentration tested to obtain the final values. Competition assays were then performed in which the protein was held at a constant concentration of 500 nM (1 μM for the FABP5NLSm palmitic acid competition), with 1,8-ANS also being held constant at either 5 μM (for hFABP5WT and hFABP5DSm) or 10 μM (for hFABP5NLSm) in the presence of increasing fatty acid concentrations from 10 nM to 200 μM. Blanks consisting of 1,8-ANS and fatty acid in the absence of protein were subtracted at each ligand concentration tested. The resulting fluorescence values were used to calculate a Ki value for the fatty acid of interest. Data were collected at 30 °C on a BioTek Synergy plate reader using an excitation filter of 380/20nm and an emission filter of 460/40nmand processed inGraphPad Prism 5. |
| Affinity data for this assay | |
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