Testing and selecting custom antibodies for two subunits of the Drosophila melanogaster mitochondrial respiratory chain complex I.

Abstract

The mitochondrial respiratory chain produces energy for the whole organism. It is divided into five different complexes named I to V. Disorders caused by mutations in the subunits of the mitochondrial complexes are severe and often lead to lethality. Drosophila melanogaster RNAi knockdown lines are used to study mitochondrial disorders, reproducing in flies symptoms observed in patients and to find out the possible gene therapy for the diseases. The RNAi knockdown lines of two subunits of complex I (CG3683 and CG6020) are used in the search of possible gene therapy. The aim of this development project is to generate custom made antibodies against these two subunits, because there are no commercial antibodies available.

The custom made polyclonal antiserums were tested and selected for affinity purification by dot blotting and western blotting. The preselection of the antibodies was done by the dot blot assay using designed peptides as antigens. The final selection of the antibodies was done by western blotting using mitochondrial proteins from Drosophila melanogaster.

In this development project high quality antibodies for subunits CG3683 and CG6020 were found. These antibodies will be used in further research of the RNAi knockdown lines. The level of the knockdown of these proteins will be determined by western blotting from whole flies and from the testes of sterile males.