Disulfide bond requirements for β2AR-T4L. (A). Ligand binding assays for β2AR-T4L expressed in vitro was performed as described in Fig. 1 using the MembraneMax Protein Expression system (Life Technologies, Carlsbad, CA) following the manufacturer's directions, which results in 1.0 mM DTT in the reaction; or using DTT-free buffers, which results in 0.2 mM DTT in the reaction; or using the RTS 100 E. coli Disulfide Kit (Roche Applied Science, Indianapolis, IN), which is designed for synthesis of disulfide-bonded proteins (detailed composition unknown). All reactions were performed in the presence of NLPs and either 0.5 nM [3H]DHA (for ligand binding determination) or [35S]Met (for protein quantity assessment). [35S]Met trace-labeled products were separated by SDS electrophoresis, and visualized by autoradiography). (B) Reactions using β2AR-T4L and two cysteine mutant derivatives were performed using the MembraneMax Protein Expression system supplemented with DTT-free buffers and processed as described in (A).