L8 ultracentrifuge

Prepore was produced by two separate methods that were published [1 (link)] [2 (link)]. Briefly, in the first method, 1 µg protoxin was mixed with 10 µg insect brush border membrane vesicles (BBMV) in the presence of 50 µl of solubilization buffer (50 mM Na₂CO₃ pH 10.5 + 0.2% β-mercaptoethanol) and incubated at room temperature (25°C – 28°C) for 15 min. In the second method, 200 ng of protoxin was incubated with scFv73 in a 1:2 mass ratio and 5% midgut juice was added in 100 µl of solubilization buffer containing 60 µM small unilamelar vesicles made of 1,2-Dioleoyl-sn-Glycero-3-phosphocholine (DOPC). The mixture was incubated at 37°C for 1 hr, followed by precipitating the membrane bound toxin at 400,000× g in a Beckman L8 ultracentrifuge. A control reaction, which lacked any small unilamellar vesicles, was used to show no precipitate formation in the absence of SUV. The pellet was resuspended in 50 µl of buffer in presence of 10% n-octyl-β-D-glucopyranoside and clarified by centrifugation, treated with loading dye and boiled for 3 – 5 min. Western blot analysis of the protein was performed using polyclonal anti-prepore antibody (1:50,000; 1hr) and secondary HRP antibody (1:10,000; 1hr) and was detected using chemiluminescence substrate (Bio-Rad).