1Institute of Organic Chemistry, Department of Chemistry, RWTH Aachen University
DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.
Published November 22, 2014. Keywords: Biochemistry, S-adenosyl-l-methionine, AdoMet, SAM, aziridine cofactor, double activated cofactor, methyltransferase, DNA methylation, protein methylation, biotin labeling, fluorescence labeling, SMILing, mTAG
1Department of Administration and Pharmaceutical Sciences, Presbyterian College School of Pharmacy
A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.
Published November 22, 2014. Keywords: Basic Protocols, Zebrafish, in vivo, novice, practical, construction, affordable, aquarium, aquaculture, fish breeder racks.
JoVE Immunology and Infection
1Department of Pathology, University of California, San Diego
This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.
Published November 21, 2014. Keywords: Immunology, T cell, immunology, human cell culture, transplantation, flow cytometry, alloreactivity
JoVE Immunology and Infection
1Department of Medicine - Division of Infectious Diseases, Vanderbilt University School of Medicine, 2Tennessee Valley Healthcare Systems, U. S. Dept. of Veterans Affairs
Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.
Published November 21, 2014. Keywords: Infection, Helicobacter pylori, iron acquisition, cag pathogenicity island, type IV secretion, pili
JoVE Immunology and Infection
1Department of Immunology, Genetics and Pathology, Uppsala University
The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).
Published November 21, 2014. Keywords: Immunology, In vitro human model system, whole blood, endothelial cells, vascular activation, inflammation, blood coagulation
1Division of Neurobiology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, 2Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, 3Centre for Integrative Physiology, University of Edinburgh, 4Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh
The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.
Published November 20, 2014. Keywords: Basic Protocols, western blotting, fluorescent, LI-COR, protein, quantitative analysis, loading control
JoVE Applied Physics
1Craft and Hawkins Department of Petroleum Engineering, Louisiana State University
This article reports on a laboratory scale investigation of an existing field procedure and its adaptation for sealing of leaky wellbores. It consists of mechanical expansion of metal pipe, which results in an improved metal/cement bond, ultimate sealing of hydraulic pathways and prevention of gas leaks caused by the presence of a microannular channel.
Published November 20, 2014. Keywords: Physics, Leaky wellbores, Wellbore cement, Microannular gas flow, Sustained casing pressure, Expandable casing technology.
1Complex Carbohydrate Research Center, University of Georgia, 2Department of Biochemistry and Molecular Biology, University of Georgia, 3Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University
In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive β-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.
Published November 20, 2014. Keywords: Chemistry, glycoprotein, glycosylation, in-gel reductive β-elimination, O-linked glycan, sulfated glycan, mass spectrometry, protein ID, SDS-PAGE, glycomics, sulfoglycomics
1Department of Psychology, McGill University, 2Department of Cognitive Science, McGill University, 3Douglas Institute, Department of Psychiatry, McGill University, 4Neurology and Neurosurgery Department, McGill University
Using event-related EEG potentials (ERPs), we investigate the effects of antipsychotic medications on abnormal semantic brain activations in healthy individuals with schizotypal traits. We use ERPs to track distinct changes in brain activity, shedding insight into the cognitive processes associated with semantic categorization.
Published November 19, 2014. Keywords: Behavior, Electrical brain activity, Semantic categorization, Event-related brain potentials, Neuroscience, Cognition, Psychiatry, Antipsychotic medication, N400, Schizotypy, Schizophrenia.
1Department of Physiology, University of California Los Angeles, 2Department of Neurobiology, University of California Los Angeles
The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.
Published November 19, 2014. Keywords: Neuroscience, astrocyte, calcium, striatum, GECI, GCaMP3, AAV2/5, stereotaxic injection, brain slice, imaging