Manduca sexta is a model insect for biochemical and physiological studies, including those involving Bacillus thuringiensis (Bt) toxins. Despite its widespread use, the protein components of M. sexta midgut are mostly unknown. Vesicles prepared from the brush border of M. sexta midgut were analyzed using one- and two-dimensional gel electrophoresis (2DE). Gels were silver stained as well as blotted to filter for Western blot analysis. Proteins separated by 2DE revealed multiple species at a given molecular size, and 2DE blots probed with biotinylated Bt Cry1Ac toxin were more complex than blots from one-dimensional separations. Vesicle proteins that were treated with PIPLC looked similar between 1DE and 2DE blots analyzed with anti-CRD antibody. Antibodies against phosphorylated amino acids also revealed multiple phosphorylated proteins. Identification of spots from 2DE gels was done using MALDI-TOF and TOF-TOF techniques. This study demonstrated that using 2DE, an assortment of proteins are revealed that are not seen in one-dimensional analyses. The 2DE technique also reveals more Cry1Ac binding epitopes in blot analyses. This study also shows the obstacles in protein identification when using organisms with unsolved genomes.
Species 1: Lepidoptera Sphingidae Manduca Sexta
Keywords: Bacillus thuringiensis, mass spectroscopy
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