Complete his resin

Oligonucleotides were purchased from Operon Biosciences. Restriction enzymes and DNA-modifying enzymes were obtained from New England Biolabs. The Complete His resin was from Roche. The purified Strep-Tactin XT, Strep-Tactin XT Superflow and MagStrep “type3” XT beads were kindly provided by IBA GmbH. Research grade sensor chip CM4, immobilization reagents NHS, EDC, and ethanolamine and HBS-N buffers for SPR experiments were from GE Healthcare.
Cholesteryl hemisuccinate Tris salt (CHS) and detergents 3 [(cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and n-dodecyl-b-D-maltosyde (DDM) were obtained from Anatrace. Lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine sodium salt (POPS) were purchased from Avanti Polar Lipids Inc. Synthetic cannabinoid ligand CP-55,940 was from Tocris. All other chemicals of reagent grade were purchased from Sigma.

Detergent-solubilized CB₂-130 and CB₂-290 proteins were purified by immobilized metal-affinity chromatography (IMAC) on a Complete His resin (Roche) as described above, and 1, 4, or 14 mL extract were mixed with 0.2 mL Strep-Tactin XT resin. In the case of the construct CB₂-293 that lacks the C-terminal His-tag, a biotin blocking solution (BioLockTM, IBA GmbH) was added to the extract according to the manufacturer’s protocol, to sequester endogenous biotin present in E. coli cells. Then either 4, 40, or 200 mL of solubilized extract were mixed with 0.2 mL resin. Incubation continued for 1 hour at 4°C. We later demonstrated that shorter incubation for 10-15 min were equally effective in capturing the protein (results not shown). The resin was collected by centrifugation, transferred into a disposable column, washed with 10 mL of buffer A + 0.5 M NaCl + CP-55,940, and protein was eluted with 4x column volumes of buffer A + 1M NaCl + CP-55,940 + 50 mM biotin.