Russian Academy of Sciences View Institution's Website 10 articles published in JoVE Biochemistry Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions Artem Bonchuk1,2, Nikolay Zolotarev1, Konstantin Balagurov1,2, Olga Arkova2, Pavel Georgiev1 1Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 2Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro. Biochemistry Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry Nadezhda Podoplelova1,2, Polina Soloveva1,4, Andrei Garzon Dasgupta1,2,3, Aleksandra Filkova1,2, Mikhail Panteleev1,2,3,4 1Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, 2National Medical Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev, 3Faculty of Physics, Lomonosov Moscow State University, 4Faculty of Biological and Medical Physics, Moscow Institute of Physics and Technology Here, we describe a set of methods for characterizing the interaction of proteins with membranes of cells or microvesicles. Biochemistry Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach Sergey I. Kovalchuk1, Rustam Ziganshin1, Irina Shelukhina2 1Laboratory of Bioinformatics Methods in Combinatorial Chemistry and Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 2Department of Molecular Neuroimmune Signalling, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Here we present a protocol for the optimized FlashPack capillary column packing procedure. Application of an optimized protocol to a common 100-bar pressure bomb setup allows 10-times faster packing and manufacturing of long ultra-high performance capillary columns. Immunology and Infection Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung Ivan V. Maslov1, Andrey O. Bogorodskiy1, Mariia V. Pavelchenko1, Ilia O. Zykov1, Natalya I. Troyanova2, Valentin I. Borshchevskiy1,3, Marina A. Shevchenko2 1Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 2Department of Immunology, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 3Institute of Biological Information Processing (IBI-7: Structural Biochemistry), Forschungszentrum Jülich We describe the method for quantitative analysis of the distribution of Aspergillus fumigatus conidia (3 µm in size) in the airways of mice. The method also can be used for the analysis of microparticles and nanoparticle agglomerate distribution in the airways in various pathological condition models. Neuroscience Event Related Potentials (ERPs) and other EEG Based Methods for Extracting Biomarkers of Brain Dysfunction: Examples from Pediatric Attention Deficit/Hyperactivity Disorder (ADHD) Geir Ogrim1,2,3, Juri D. Kropotov4,5 1Neuropsychiatric Team, Åsebråten Outpatient Clinic, Østfold Hospital Trust, 2Institute of Psychology, Norwegian University of Science and Technology, 3Gillberg Neuropsychiatry Centre, University of Gothenburg, 4P. Bechtereva Institute of the Human Brain, Russian Academy of Sciences, 5Department of Neuropsychology, Andrzej Frycz-Modrzewski Krakow University EEG-methods are applied for extracting biomarkers of brain dysfunctions. The focus is on multi-channel event-related potentials (ERPs) recorded in a cued GO/NOGO task. Non-brain artifacts are corrected and ERPs are compared with the normative data. Examples relate to biomarkers for ADHD diagnosis and prediction of medication response. Immunology and Infection Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals Sara Olivera-Ardid*1, Nailya Khasbiullina*2,3, Alexey Nokel3, Andrey Formanovsky2, Inna Popova2, Tatiana Tyrtysh2, Roman Kunetskiy2, Nadezhda Shilova2, Nicolai Bovin2,4, Daniel Bello-Gil1, Rafael Mañez1,5 1Infectious Pathology and Transplantation Division, Bellvitge Biomedical Research Institute (IDIBELL), 2Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry (IBCh), Russian Academy of Sciences, 3Semiotik LLC, 4Auckland University of Technology, 5Intensive Care Department, Bellvitge University Hospital This work shows the potential of printed glycan array (PGA) technology for the analysis of circulating anti-carbohydrate antibodies in small animals. Biochemistry Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis Tomáš Zavřel1, Dominik Chmelík1,2, Maria A. Sinetova3, Jan Červený1 1Department of Adaptive Biotechnologies, Global Change Research Institute, Czech Academy of Sciences, 2Department of Plant Physiology, Faculty of Science, Masaryk University, 3Laboratory of Intracellular Regulation, Institute of Plant Physiology, Russian Academy of Sciences Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually. Neuroscience Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction Dmitry V. Samigullin1,2,3, Eduard F. Khaziev1,2,3, Nikita V. Zhilyakov1,2, Ellya A. Bukharaeva1,2, Eugeny E. Nikolsky1,2,4 1Laboratory of Biophysics of Synaptic Processes, Kazan Scientific Centre, Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, 2Open Laboratory of Neuropharmacology, Kazan Federal University, 3Department of Radiophotonics and Microwave Technologies, A.N. Tupolev Kazan National Research Technical University, 4Department of Medical and Biological Physics, Kazan State Medical University Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings. Biology Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends Vladimir A. Volkov1,2, Anatoly V. Zaytsev3, Ekaterina L. Grishchuk3 1Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, 2Federal Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia, 3Physiology Department, Perelman School of Medicine, University of Pennsylvania Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends. Biology Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo Benjamen A. Filas1, Victor D. Varner1, Dmitry A. Voronov1,2, Larry A. Taber1,3 1Department of Biomedical Engineering, Washington University, 2Institute for Information Transmission Problems, Russian Academy of Sciences, 3Department of Mechanical Engineering and Materials Science, Washington University This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.
