NPC1L1 Cholesterol Binding Assay

80 µl solutions containing His-tagged NPC1L1 N-terminal domain or Domain2 (150 nM) were incubated with cholesterol (5 µM, 1:1000 ³H:¹H) in Buffer A (50 mM HEPES (pH 7.4), 150 mM NaCl), containing either 0.004% NP-40 and BSA (192 nM) or mixed bile salt micelles for 30 min at 37°C. Micelle lipids were first dissolved in ethanol, combined in a glass vial, and solvents evaporated under N₂. The dried lipids were incorporated into buffer A containing 24 mM sodium taurocholate by vortexing for 5 min. The solution was then diluted using buffer A to final concentrations: 5 mM sodium taurocholate, 0.5 mM oleic acid, 0.035 mM phosphatidylcholine, 0.08 mM lysophosphatidylcholine, 0.3 mM monoolein, 0.005 mM cholesterol, and 5 nM radiolabeled cholesterol (Johnson and Pfeffer, 2016 (link)). After incubation, solutions were loaded onto 1 ml syringes fitted with a frit and 20 µl Ni-NTA resin. After 15 min, the resin was washed 6X with 1 ml wash buffer (buffer A + 10 mM imidazole + 0.004% NP-40) and eluted with 1.2 ml elution buffer (buffer A + 250 mM imidazole + 0.004% NP-40). Eluted samples were mixed with BioSafe-II (Research Products International, Mt. Prospect, IL), and radioactivity was determined using a scintillation counter.