| Assay Method Information | |
| | Sterol-Binding Assay |
| Description: | Recombinant OSBP, ORP4L, or ORP4S (8 pmol) was incubated in 75 μl of binding buffer (10 mM HEPES (pH 7.4), 150 mM KCl, 2% (w/v) polyvinyl alcohol) containing radioactive sterols (with or without a 40-fold excess of unlabeled sterol). After 2 h at 20 °C, a 25-μl slurry of Talon resin (1:1, v/v) was added and mixed for 10 min. Talon resin and bound protein were washed three times with 300 μl of 10 mM HEPES (pH 7.4) and 150 mM KCl and sedimented by brief centrifugation. Bound proteins were eluted from the resin with 150 mM imidazole (pH 7.4), the resin was sedimented by centrifugation, and radioactivity bound to protein in the supernatant was measured by liquid scintillation counting. |
| Affinity data for this assay | |
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