Manduca sexta is a model insect for physiological and biochemical studies, including those involving Bacillus thuringiensis (Bt) toxins. Despite widespread use of midgut from M. sexta, it's protein components are mostly unknown. Vesicles prepared from the brush border of M. sexta midgut were analyzed using traditional one- and two-dimensional gel electrophoresis (2DE). Gels were silver stained, as well as blotted to filters for analysis using biotinylated Bt Cry1Ac toxin. Proteins separated by 2DE revealed multiple species at a given molecular size, and blots probed with Cry1Ac toxin were more complex than visualized with one-dimensional separations. Midgut vesicle proteins were digested with PIPLC to cleave GPI-anchored proteins. Gels from these digests were silver stained and also transferred to filters for analysis with anti-CRD antibody and Bt toxin. N-terminal sequencing of selected protein spots identified several proteins. This study demonstrates that using 2DE, an assortment of proteins are revealed that are not seen on traditional one-dimensional gel analyses. The 2DE technique also reveals more Cry1Ac binding epitopes in blot analyses.
Species 1: Lepidoptera Manduca sexta
Keywords: Bacillus thuringiensis, membrane
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