Thermowell pcr tubes

Purified Bpa incorporated REEP/Yop1 mutants (5 μM) in buffer containing 0.03% DDM or reconstituted into liposomes generated with a S. cerevisiae lipid extract at a protein:lipid molar ratio of 1:200 were added to Thermowell PCR tubes (Corning) and placed on an ice-cold metal block. A long-wave UV lamp (Blak-Ray) was positioned 5 cm from the tubes, and the samples were irradiated for 30 min. After irradiation, SDS sample buffer was added. The samples were then subjected to SDS-PAGE and Coomassie-blue staining.

To incorporate photoreactive Bpa probes into sjRop1, amber codons were introduced into Rop1 at various positions (see Supplementary Table 2 for plasmids). The mutated Rop1 expression plasmid and the amber suppressor tRNA plasmid were co-transformed into BL21 (DE3) cells (NEB), as described^{5 (link)}. One mM final concentration of H-Bpa-OH (Chempep) was added to the medium, and the amber suppressor tRNA was induced 1–2 h before induction of sjRop1 expression.
For photo-crosslinking, 20 µl of a membrane suspension in 25 mM HEPES/KOH pH 7.5, 150 mM NaCl was added to Thermowell PCR tubes (Corning) and placed onto an ice-cold metal block. The samples were irradiated for 30 min with a long-wave UV lamp (Blak-Ray) and then analyzed by SDS-PAGE and Western blotting with DyLight 800-labeled streptavidin (Invitrogen) diluted to 1:4000 in 1% BSA.