Deoxy bigchap

SecYEG was reconstituted into E. coli polar lipid extract (Avanti Polar Lipids, Alabaster, AL, USA) vesicles pre-dissolved in deoxy-BigChap (Anatrace, Maumee, OH, USA) as previously described [3 (link)]. Biobeads SM2 (Biorad, Hercules, CA, USA) were added to remove the excess detergent and the resulting turbid suspension was pelleted at 100.000 g. The resulting pellet was resuspended and extruded through a 100 nm filter. Mass ratios of protein to lipid of 1:54 to 1:108 were used.

Translation reactions (100 μl) were programed with mRNA templates encoding an N-terminal 3x-FLAG tag or 3x-HA tag (control) followed by the first 126 residues of TNFα. Reactions were assembled in the presence or absence of CT7 (1 μM). Following translation, ER microsome-targeted RNCs were isolated as described above, except the membrane pellet was brought up in two volumes of membrane buffer supplemented with 1% Deoxy BigChap (DBC, Anatrace, Santa Clara, CA). The membranes were solubilized for 10 min at 0°C and insoluble material removed by centrifugation at 50,000 rpm in a TLA100 rotor at 4°C for 10 min. The supernatant was then incubated with anti-FLAG affinity resin and mixed with rotation at 4°C for 2.5 hr. The resin was then sedimented (600 × g, 3 min, 4°C) and washed four times with 1 ml of membrane buffer containing 0.3% DBC. After the final wash, bound proteins were eluted with 250 μg/ml of 3x-FLAG peptide (Sigma) in membrane buffer containing 0.3% DBC. The eluted material was analyzed directly by SDS-PAGE and semi-quantitative western blotting, or was first photolyzed and then subjected to click chemistry and analyzed by SDS-PAGE and in-gel fluorescent scanning. Typically, ∼0.3 pmol of purified RNC-Sec61 complexes were obtained from a 100 μl translation reaction.