Stowers Institute for Medical Research View Institution's Website 7 articles published in JoVE Biology Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry Shriram Venkatesan*1, Tejbir S. Kandola*1, Alejandro Rodríguez-Gama1, Andrew Box1, Randal Halfmann1,2 1Stowers Institute for Medical Research, 2Department of Molecular and Integrative Physiology, The University of Kansas School of Medicine This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells. Biology Gamete Collection and In Vitro Fertilization of Astyanax mexicanus Robert Peuß*1, Zachary Zakibe*1, Jaya Krishnan1, M. Shane Merryman1, Diana P. Baumann1, Nicolas Rohner1,2 1Stowers Institute for Medical Research, 2Department of Molecular & Integrative Physiology, KU Medical Center In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus. Genetics Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events Melvin Noe Gonzalez1, Joan W. Conaway1,2, Ronald C. Conaway1,2 1Stowers Institute for Medical Research, 2Department of Biochemistry and Molecular Biology, Kansas University Medical Center Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex. Biology Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes Lu Chen1,2, Soon-Keat Ooi1, Joan W. Conaway1,2, Ronald C. Conaway1,2 1Stowers Institute for Medical Research, 2Department of Biochemistry & Molecular Biology, Kansas University Medical Center Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner. Biology Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes Lu Chen1,2, Soon-Keat Ooi1, Ronald C. Conaway1,2, Joan W. Conaway1,2 1Stowers Institute for Medical Research, 2Department of Biochemistry & Molecular Biology, Kansas University Medical Center This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography. Biology Planarian Immobilization, Partial Irradiation, and Tissue Transplantation Otto C. Guedelhoefer IV1,2, Alejandro Sánchez Alvarado3,4 1Department of Neurobiology and Anatomy, University of Utah School of Medicine, 2Department of Molecular, Cellular and Developmental Biology, UCSB, 3Howard Hughes Medical Institute, 4Stowers Institute for Medical Research An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals. Neuroscience Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor Limei Ma1, Sachiko Haga-Yamanaka1, Qingfeng Elden Yu1, Qiang Qiu1, SangSeong Kim1, C. Ron Yu1,2 1Stowers Institute for Medical Research, 2Department of Anatomy and Cell Biology, The University of Kansas School of Medicine The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.
