MRC Laboratory of Molecular Biology 8 articles published in JoVE Biology Imaging ATG9A, a Multi-Spanning Membrane Protein Alexander R. van Vliet*1,2, Stefano De Tito*1, Eugenia Almacellas*1, Sharon A. Tooze1 1Molecular Cell Biology of Autophagy, The Francis Crick Institute, 2MRC Laboratory of Molecular Biology This protocol describes various methods that can help in the study of ATG9A biology, including immunofluorescence followed by image analysis, transient overexpression considerations, and investigating the ATG9A glycosylation status using western blot. Genetics Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria Peggy Paschke1, David A. Knecht2, Thomas D. Williams1, Peter A. Thomason3, Robert H. Insall3, Jonathan R. Chubb4,5, Robert R. Kay1, Douwe M. Veltman1 1MRC Laboratory of Molecular Biology, 2Department of Molecular and Cell Biology, University of Connecticut, 3Cancer Research UK Beatson Institute Glasgow, 4MRC Laboratory for Molecular Cell Biology, University College London, 5Department of Cell and Developmental Biology, University College London Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media. Chemistry Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction Benjamí Oller-Salvia1 1Medical Research Council Laboratory of Molecular Biology Incorporating a cyclopropene derivative of lysine into antibodies allows the site-specific, rapid and efficient linkage of tetrazine-bearing molecules to generate antibody-drug conjugates. Behavior A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice Ruben Vale1,2, Dominic Evans1,2, Tiago Branco1,2 1UCL Sainsbury Wellcome Centre for Neural Circuits and Behaviour, 2MRC Laboratory of Molecular Biology This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment. Biochemistry Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology Fabrice Gorrec1, Jan Löwe1 1Laboratory of Molecular Biology, Medical Research Council Automated systems and protocols for the routine preparation of a large number of screens and nanoliter crystallization droplets for vapor diffusion experiments are described and discussed. Biology Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR) Priya Crosby1, Nathaniel P. Hoyle1, John S. O'Neill1 1MRC Laboratory of Molecular Biology Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research. Biology Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli Louise E. Bird1, Heather Rada1, Anil Verma1, Raphael Gasper2,3, James Birch4, Matthew Jennions4, Jan Lӧwe3, Isabel Moraes4, Raymond J. Owens1 1Oxford Protein Production Facility, Research Complex at Harwell, 2Protein Crystallography Group, Ruhr University, 3MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, 4Membrane Protein Laboratory, Diamond Light Source A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented. Neuroscience Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun Jason Arsenault1,2, Andras Nagy1, Jeffrey T. Henderson1, John A. O'Brien2 1Leslie Dan Faculty of Pharmacy, University of Toronto, 2MRC-Laboratory of Molecular Biology, Cambridge, UK Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.