Investigations into the use of capillary electrophoresis for the analysis of nuclear preparations and the nuclear localization of fusion proteins at the single cell level

This thesis describes the development and application of new capillary electrophoretic (CE) methods for the analysis of nuclei in bulk preparations isolated from cell cultures, as well as from single cells. The bulk level analyses are used to monitor “purity” of nuclear preparations. The single cell studies are used to analyze the localization of a nuclear targeted fusion protein. Furthermore, they are used to determine the nuclear localization of aminoacyl-tRNA synthetases (aaRSs), a key enzyme family involved in protein translation.; First, a new CE method was developed that established the capability of CE to separate and detect individual species from bulk nuclear preparations. Using this bulk method it was shown that nuclear preparations are contaminated by fragmented nuclei, mitochondria and cytoskeletal remnants.; The feasibility of analyzing nuclei from single cells using CE-LIF was demonstrated using cells expressing a nuclear targeted fusion protein, nuDsRed. This study highlights the use of CE to study the nuclear localization of fusion proteins that are expressed at low levels due to low transfection efficiencies. Finally, using this single cell method, methionyl-tRNA synthetase, expressed as an enhanced green fluorescent protein (EGFP) labeled fusion protein, was localized both in the cytoplasm and the nuclei of human osteosarcoma cells, while lysyl-tRNA synthetase, similarly expressed, was only detected in the cytoplasm.