title: Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions.
abstract: A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides  mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.
authors.affiliation: Institute for Biochemistry, Emil-Fischer Centre, University of Erlangen-Nuremberg.
authors.forename: Kristin
authors.initials: K
authors.lastname: Derlig
authors.affiliation: Department of Biology, Animal Physiology, University of Erlangen-Nuremberg.
authors.forename: Andreas
authors.initials: A
authors.lastname: Gießl
authors.affiliation: Department of Biology, Animal Physiology, University of Erlangen-Nuremberg.
authors.forename: Johann Helmut
authors.initials: JH
authors.lastname: Brandstätter
authors.affiliation: Institute for Biochemistry, Emil-Fischer Centre, University of Erlangen-Nuremberg.
authors.forename: Ralf
authors.initials: R
authors.lastname: Enz
authors.affiliation: Institute for Biochemistry, Emil-Fischer Centre, University of Erlangen-Nuremberg; rdahlhaus@hotmail.com.
authors.forename: Regina
authors.initials: R
authors.lastname: Dahlhaus
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003543:Cysteamine / Q000031:analogs & derivatives* / Q000378:metabolism; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; D009687:Nuclear Proteins; C503924:Pep-1 peptide; D010455:Peptides; D003543:Cysteamine; D012313:RNA