Purification of Timolol-Bound GPCR Receptors

Frozen membranes were thawed on ice and NaCl, MgCl₂, and GTPγS were added to reach final concentrations of 300 mM, 1 mM, and 10 μM, respectively. Timolol was then added to a final concentration of 1 μM and the membranes were incubated for 10 minutes on ice. Receptors were solubilized for 1 hour at 4 °C in the presence of 1% dodecylmaltoside (DDM) and 0.1% cholesterol hemisuccinate (CHS). Following centrifugation for 30 minutes at 25,000g, the supernatant was applied to Ni-NTA agarose. The column was slowly washed with 20 column volumes of 20 mM HEPES, pH 8.0, 300 mM NaCl, 0.1% DDM, 0.01% CHS to remove bound timolol. Receptor was eluted in the same buffer plus 200 mM imidazole and concentrated using an Amicon 30 kDa-cutoff spin concentrator for addition to the rHDL reconstitution mixture.