Universitatsmedizin Gottingen 10 articles published in JoVE Biochemistry Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy Tanja D. Becke1,2,3, Stefan Ness2, Stefanie Sudhop1,3, Hermann E. Gaub3, Markus Hilleringmann2, Arndt F. Schilling*4, Hauke Clausen-Schaumann*1,3 1Center for Applied Tissue Engineering and Regenerative Medicine, Munich University of Applied Sciences, 2FG Protein Biochemistry & Cellular Microbiology, Munich University of Applied Sciences, 3Center for Nano Science, Ludwig-Maximilians-Universität München, 4Klinik für Unfallchirurgie, Orthopädie und Plastische Chirurgie, University Medical Center Göttingen This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin. Neuroscience An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins Donatus Riemann1, Andoniya Petkova1, Thomas Dresbach1, Rebecca Wallrafen1 1Institute for Anatomy and Embryology, University Medical Centre Göttingen We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest. Developmental Biology Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration Christine M. Rostosky1,2, Ira Milosevic1,2 1European Neuroscience Institute (ENI), 2University Medical Center Göttigen (UMG) In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines). Cancer Research Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma Jan Hagemann*1, Christian Jacobi*2, Sabine Gstoettner3, Christian Welz4, Sabina Schwenk-Zieger3, Roland Stauber1, Sebastian Strieth1, Julian Kuenzel1, Philipp Baumeister3, Sven Becker1,3 1Department of Otorhinolaryngology, Johannes-Gutenberg University Medical Center, 2Department of Otorhinolaryngology, Technical University of Munich Medical Center, 3Department of Otorhinolaryngology, Ludwig-Maximilian-University Medical Center, 4Department of Otorhinolaryngology, University of Goettingen Medical Center We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future. Neuroscience Optimized Management of Endovascular Treatment for Acute Ischemic Stroke Katharina Schregel1,2, Daniel Behme1, Ioannis Tsogkas1, Michael Knauth1, Ilko Maier3, André Karch4, Rafael Mikolajczyk4,5, Mathias Bähr3, Jörn Schäper6, José Hinz6, Jan Liman3, Marios-Nikos Psychogios1 1Institute of Neuroradiology, University Medical Center Goettingen, 2 The outcome of patients with acute ischemic stroke depends on swift restoration of cerebral blood flow. This protocol aims at optimizing the management of such patients by minimizing peri-procedural timings and rendering the time from hospital admission to reperfusion as short as possible. Behavior Simultaneous Transcranial Alternating Current Stimulation and Functional Magnetic Resonance Imaging Kathleen A. Williams1, Yuranny Cabral-Calderin1,3, Carsten Schmidt-Samoa1, Christiane Anne Weinrich1,2, Peter Dechent1, Melanie Wilke1,3,4 1Department of Cognitive Neurology, University Medicine Goettingen, 2Department of Neurology, University Medicine Goettingen, 3German Primate Center, Leibniz Institute for Primate Research, 4DFG Center for Nanoscale Microscopy & Molecular Physiology of the Brain (CNMPB) Transcranial alternating current stimulation (tACS) is a promising tool for noninvasive investigation of brain oscillations, though its effects are not completely understood. This article describes a safe and reliable setup for applying tACS simultaneously with functional magnetic resonance imaging, which can increase understanding oscillatory brain function and effects of tACS. Biology Isolation and Characterization of Microvesicles from Peripheral Blood Kerstin Menck1, Annalen Bleckmann1, Matthias Schulz1, Lena Ries1, Claudia Binder1 1Department of Hematology/Medical Oncology, University Medical Center Göttingen Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting. Genetics Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP) Andreas E. Zautner1, Raimond Lugert1, Wycliffe O. Masanta1, Michael Weig1, Uwe Groß1, Oliver Bader1 1Institut für Medizinische Mikrobiologie, Universitätsmedizin Göttingen Mass spectrometry-based phyloproteomics (MSPP) was used to type a collection of Campylobacter jejuni ssp. doylei isolates at the strain level in comparison to multilocus sequence typing (MLST). 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. Neuroscience The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo Thomas Hassenklöver1, Ivan Manzini1 1Institute of Neurophysiology and Cellular Biophysics and Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), University of Göttingen We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.
