Molecular typhoon phosphoimager

The binding affinities of the different RNA pools or individual aptamers were determined by nitrocellulose-membrane filtration-based saturation binding assay. Constant amounts of 5′-[³²P]-radiolabelled RNA aptamers (at 2000 CPM/µL final) were incubated with increasing concentrations of the β₂AR or β₂AR:BI (12 two-fold serial dilutions starting from 2 µM) in a buffer containing 20 mM HEPES, pH 7, 50 mM NaCl, 2 mM MgCl₂, 2 mM CaCl₂, 0.01% MNG and 0.001% CHS for 30 minutes at RT. The final reaction volume was 20 µL. The β₂AR-RNA aptamer mixtures were then passed through a stack of membranes on a vacuum-manifold consisting of a Protran-nitrocellulose that captures RNA-protein complexes and GeneScreen Plus® nylon membrane that captures unbound RNA molecules. After washing twice with 100 µL binding buffer the membranes were air dried for 5 minutes, exposed to Phosphoimager screens (1 hr), and scanned using a Molecular Typhoon Phosphoimager (GE Healthcare). Finally, the fraction of RNA-bound was calculated, adjusted for background and graphed using GraphPad Prism. The equilibrium dissociation constant (K_d) for the RNA aptamers were obtained by fitting the fraction of nitrocellulose-bound RNA to the following equation: Y = (Bmax*X) / (X + K_d), where Bmax is the maximum value of Y (when X = ∞); and K_d, the dissociation constant, is the value of X when Y = Bmax /2.