| Assay Method Information | |
| | Scintillation Proximity (SPA) Assay |
| Description: | In vitro binding of compounds to RBP4. Compound binding to RBP4 was assessed in the radiometric scintillation proximity (SPA) assay that was previously described (Cioffi, C. L. et al. 2014; Cioffi, C. L. et al. 2015; Cioffi, C. L. et al. 2020). The assay measured competitive displacement of radiolabeled [3H]-all-trans-retinol from native RBP4 purified from human urine (Fitzgerald, 30R-AR022L). The protein was biotinylated using the EZ-link Sulfo-NHS-LC-Biotinylation kit from ThermoFisher (Cat #21335) as recommended by the manufacturer. Binding assays were implemented in a final volume of 100 μL in SPA buffer (1×PBR, pH 7.4, 1 mM EDTA, 0.1% BRA, 0.5% CHAPS). The assay reaction included a radioligand, 10 nM [3H]-all-trans-retinol (48.7 Ci/mmol; PerkinElmer, Waltham, MA), along with the 0.3 mg/well Streptavidin-PVT beads (PerkinElmer, RPNQ0006) and 50 nM biotinylated human RBP4. Unlabeled retinol (Sigma, cat # 95144) at 20 μM was added to control wells to assess a nonspecific binding. Radioactivity counts were measured using CHAMELEON plate reader (Hidex Oy, Turku, Finland) after 16 h of incubation at rt with mild shaking. |
| Affinity data for this assay | |
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