Goethe University Frankfurt View Institution's Website 14 articles published in JoVE Biochemistry Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs Roman Levin1, Zoe Koeck1, Volker Dötsch1, Frank Bernhard1 1Institute of Biophysical Chemistry, Goethe University Frankfurt Co-translational insertion into pre-formed nanodiscs makes it possible to study cell-free synthesized membrane proteins in defined lipid environments without contact with detergents. This protocol describes the preparation of essential system components and the critical parameters for improving expression efficiency and sample quality. Biology Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes Julia Ratke*1, Franziska Krämer*1, Frederic Strobl1 1Physical Biology/Physikalische Biologie (IZN, FB15), Buchmann Institute for Molecular Life Sciences (BMLS), Cluster of Excellence Frankfurt, Macromolecular Complexes (CEF - MC), Goethe-Universität Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue pro-actively. Bioengineering Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro Liudmila Leppik1, Mit B. Bhavsar1, Karla M.C. Oliveira1, Maria Eischen-Loges1, Sahba Mobini1,2, John H. Barker1 1Frankfurt Initiative for Regenerative Medicine, Johann Wolfgang Goethe-University, 2J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation. Biochemistry Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids Kerstin Pieper1, Kathi Gundermann1, Lars Dietzel1 1Institute for Molecular Biosciences, Goethe University Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes. Chemistry Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers Martin Pitzer1,2, Kilian Fehre1, Maksim Kunitski1, Till Jahnke1, Lothar Schmidt1, Horst Schmidt-Böcking1, Reinhard Dörner1, Markus Schöffler1 1Institut für Kernphysik, Goethe-Universität Frankfurt am Main, 2Experimentalphysik IV, Universität Kassel For small chiral species, Coulomb Explosion Imaging provides a new approach to determine the handedness of individual molecules. Developmental Biology Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos Frederic Strobl1,2,3, Selina Klees1,2,3, Ernst H. K. Stelzer1,2,3 1Physical Biology, Buchmann Institute for Molecular Life Sciences (BMLS), 2Cluster of Excellence Frankfurt, Macromolecular Complexes, 3Goethe-Universität Frankfurt am Main – Campus Riedberg Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations. Immunology and Infection Flow Cytometry-based Assay for the Monitoring of NK Cell Functions Sara Tognarelli*1,2, Benedikt Jacobs*3,4, Nina Staiger1,2, Evelyn Ullrich1,2 1Childrens Hospital, Department of Pediatric Stem Cell Transplantation and Immunology, Johann Wolfgang Goethe-University, 2LOEWE Center for Cell and Gene Therapy, Johann Wolfgang Goethe-University, 3Institute for Cancer Research, Department of Cancer Immunology, Oslo University Hospital, Radiumhospital, 4The KG Jebsen Center for Cancer Immunotherapy, Institute of Clinical Medicine, University of Oslo A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets. Biology Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution Michael Urban1, Marc Vor der Brüggen2, Robert Tampé1 1Institute of Biochemistry, Biocenter, Goethe University Frankfurt, 2Nanospot GmbH The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings. Biology Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools Priyanka Dutta*1, Christina Lehmann*1, Devang Odedra*1, Deepika Singh*1, Christian Pohl1 1Buchmann Institute for Molecular Life Sciences and Institute of Biochemistry II, School of Medicine, Goethe University Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow. Behavior The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test Johanna U. Frisch1, Jan A. Häusser1, Rolf van Dick2, Andreas Mojzisch1 1Institute of Psychology, University of Hildesheim, 2Department of Psychology and Sports Science and Center for Leadership and Behavior in Organizations, Goethe-University Frankfurt Previous research on the social dimension of stress has focused on two important variables: social identity and social support. This protocol introduces an effective experimental manipulation of these two social variables and describes their implementation in a standard stress induction paradigm (Trier Social Stress Test). Neuroscience Custom-made Microdialysis Probe Design Jana Lietsche1, Jan Gorka2, Stefanie Hardt3, Michael Karas2, Jochen Klein1 1Department of Pharmacology, Goethe University of Frankfurt, 2Department of Pharmaceutical Chemistry, Goethe University of Frankfurt, 3Department of Clinical Pharmacology, Goethe University of Frankfurt Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand.In this work a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10. Medicine Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors Hanibal Bohnenberger1, Philipp Ströbel1, Sebastian Mohr2, Jasmin Corso3, Tobias Berg2, Henning Urlaub3,4, Christof Lenz3,4, Hubert Serve2,5,6, Thomas Oellerich2,5,6 1Institute of Pathology, University Medical Center, Göttingen, 2Department of Hematology/Oncology, Goethe University of Frankfurt, 3Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, 4Bioanalytics, Institute of Clinical Chemistry, University Medical Center, Göttingen, 5German Cancer Consortium, 6German Cancer Research Center In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry. Biology Introduction to Solid Supported Membrane Based Electrophysiology Andre Bazzone1, Wagner Steuer Costa2, Markus Braner1, Octavian Călinescu1, Lina Hatahet1, Klaus Fendler1 1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, 2Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters. Medicine The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice Edith Hintermann1, Janine Ehser1, Urs Christen1 1Pharmazentrum Frankfurt / ZAFES, Goethe University Hospital Frankfurt Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.