Max Planck Institute of Biochemistry View Institution's Website 8 articles published in JoVE Biochemistry Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics Frederik Steiert1,2, Tamara Heermann1, Nikolas Hundt3, Petra Schwille1 1Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, 2Department of Physics, Technical University Munich, 3Department of Cellular Physiology, Biomedical Center (BMC), Ludwig-Maximilians-Universität München This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls. Bioengineering Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles Yashar Bashirzadeh*1, Nadab Wubshet*1, Thomas Litschel2, Petra Schwille3, Allen P. Liu1,4,5,6 1Department of Mechanical Engineering, University of Michigan, Ann Arbor, 2John A. Paulson School of Engineering and Applied Sciences, Harvard University, 3Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, 5Department of Biophysics, University of Michigan, Ann Arbor, 6Cellular and Molecular Biology Program, University of Michigan, Ann Arbor This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions. Biology Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography Anna Bieber*1, Cristina Capitanio*1, Florian Wilfling1,2, Jürgen Plitzko1, Philipp S. Erdmann1,3 1Max Planck Institute of Biochemistry, 2Max Planck Institute for Biophysics, 3Fondazione Human Technopole Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells. Biochemistry In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers Beatrice Ramm*1, Philipp Glock*1, Petra Schwille1 1Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement. Chemistry Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography Yannig Gicquel*1,2, Robin Schubert*3,4,5, Svetlana Kapis3, Gleb Bourenkov6, Thomas Schneider6, Markus Perbandt3,4, Christian Betzel3,4,5, Henry N. Chapman1,2,4, Michael Heymann1,7 1Center for Free Electron Laser Science, DESY, 2Department of Physics, University of Hamburg, 3Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, 4The Hamburg Center for Ultrafast Imaging, University of Hamburg, 5Integrated Biology Infrastructure Life-Science Facility at the European XFEL (XBI), 6European Molecular Biology Laboratory, EMBL c/o DESY, 7Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data. Developmental Biology In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae Sandra B. Lemke1, Frank Schnorrer1,2 1Muscle Dynamics Group, Max Planck Institute of Biochemistry, 2Aix Marseille University, CNRS, IBDM Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae. Biology Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue Jacek R. Wiśniewski1 1Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue. Bioengineering Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy Felix Spira1, Julia Dominguez-Escobar1, Nikola Müller1,2, Roland Wedlich-Söldner1 1AG Cellular Dynamics and Cell Patterning, Max Planck Institute of Biochemistry, 2Helmholtz Zentrum München Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.