1Institut Curie, Centre de Recherche, CNRS, UMR 168, PhysicoChimie Curie, Université Pierre et Marie Curie, 2Kavli Institute for Brain and Mind, University of California, San Diego, 3Molecular Physiology and Biophysics Section, National Institute for Neurological Disorders and Stroke, National Institute of Health
The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods — electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.
Published January 22, 2015. Keywords: Biochemistry, Biomimetic model system, Giant Unilamellar Vesicle, reconstitution, ion channel, transmembrane protein, KvAP, electroformation, gel assisted swelling, agarose, inside-out patch clamp, electrophysiology, fluorescence microscopy
1Department of Pharmacology and Toxicology, Michigan State University, 2Department of Pharmacology, University of Vermont College of Medicine
This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.
Published January 19, 2015. Keywords: Biochemistry, smooth muscle function, receptor pharmacology, signal transduction, tissue bath, rat, aorta, aortic rings, isometric contraction, concentration response curve
1Department of Medicine, Keck School of Medicine, University of Southern California
We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express α-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.
Published January 18, 2015. Keywords: Cellular Biology, Cellular biology, mouse, human, esophagus, mesenchymal stromal cells, myofibroblasts, primary cells
1Research Group Experimental Surgery, Department of Cardiovascular Surgery, Medical Faculty, Heinrich-Heine-University Dusseldorf
A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.
Published January 17, 2015. Keywords: Biochemistry, Exosome, exosome separation, microvesicle, nanoparticle-tracking analysis, nanoparticle counting, particle size analysis, ultracentrifugation, exosome isolation, blood
1Department of Entomology, Virginia Tech
A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.
Published January 16, 2015. Keywords: Infectious Diseases, Anopheles gambiae, malaria mosquito, DZNep, SAH, toxicological assay, epigenetics, vector control
1Department of Physiology and Biophysics, Virginia Commonwealth University
This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.
Published January 9, 2015. Keywords: Structural Biology, 3D electron microscopy, cryo-electron microscopy, membrane proteins, ryanodine receptor, single particle image processing, transmission electron microscopy
1Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University
Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.
Published January 8, 2015. Keywords: Cellular Biology, Caenorhabditis elegans, neurodegenerative diseases, protein misfolding diseases, prion-like spreading, cell-to-cell transmission, protein aggregation, non-cell autonomous toxicity, proteostasis
1Department of Biochemistry, University of Leicester, 2Center for Fisheries, Environment and Aquaculture Sciences, 3ES Cell Facility, Centre for Core Biotechnology Services, University of Leicester
Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term ‘subcloning plus insertion’, which can be used to generate custom gene targeting vectors rapidly.
Published January 8, 2015. Keywords: Molecular Biology, recombineering, gap-repair, subcloning plus insertion, transgene, knockout, mouse
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.
Published January 6, 2015. Keywords: Microbiology, membrane proteins, green fluorescent protein, fluorescence detection, Escherichia coli, expression screening
1Department of Biology, University of Rochester, 2W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges
Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.
Published January 6, 2015. Keywords: Genetics, Fluorescence in situ hybridization, Drosophila, Nasonia, heterochromatin, satellite DNA, chromosomes