Skip to main content
. Author manuscript; available in PMC: 2012 Jun 29.
Published in final edited form as: Biochemistry. 2010 Dec 29;50(3):388–396. doi: 10.1021/bi101239r

FIGURE 4. Analysis of interaction of N-terminal or C-terminal fragments with the membrane.

FIGURE 4

Panel A, C-terminal fragment was incubated with SUV (PC:Ch:S mixture) then the membrane fraction was separated by centrifugation at 90,000 rpm for 30 min and visualized by Western blot using polyclonal anti-Cyt1Aa/11 antibody and a secondary Goat-HRP antibody. Panel B, interaction of N terminal fragment with SUV, conditions were identical to Panel A. Panel C, effect of monoclonal antibody 10B9 at 3:1 molar ratio (10B9:Cyt1Aa) on the binding of C-terminal fragment to SUV liposomes. The presence of C-terminal fragment bound to the membrane was visualized by Western blot as described for panel A. Panel D, binding competitions assay analyzing the binding of biotin-labeled Cyt1Aa toxin to SUV liposomes in the presence of 100 fold or 1000 fold molar excess of unlabeled C- or N- terminal fragments. The bioninylated toxin was visualized with streptavidin-HRP conjugate Scanning optical density of the 25 kDa bands in the blot was performed to quantify binding. Size of proteins was estimated from molecular pre-stained precision plus standard, all blue (BioRad). S, supernatant and P, membrane pellet.