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JoVE Biology
 JoVE Biology

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

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


JoVE 52281

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.

 JoVE Biology

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

1Department of Pharmacology and Toxicology, Michigan State University, 2Department of Pharmacology, University of Vermont College of Medicine


JoVE 52324

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.

 JoVE Biology

Isolation of Myofibroblasts from Mouse and Human Esophagus

1Department of Medicine, Keck School of Medicine, University of Southern California


JoVE 52215

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.

 JoVE Biology

An Innovative Method for Exosome Quantification and Size Measurement

1Research Group Experimental Surgery, Department of Cardiovascular Surgery, Medical Faculty, Heinrich-Heine-University Dusseldorf


JoVE 50974

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.

 JoVE Biology

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

1Department of Entomology, Virginia Tech


JoVE 52041

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.

 JoVE Biology

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

1Department of Physiology and Biophysics, Virginia Commonwealth University


JoVE 52311

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.

 JoVE Biology

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

1Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University


JoVE 52321

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.

 JoVE Biology

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

1Department of Biochemistry, University of Leicester, 2Center for Fisheries, Environment and Aquaculture Sciences, 3ES Cell Facility, Centre for Core Biotechnology Services, University of Leicester


JoVE 52155

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.

 JoVE Biology

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

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


JoVE 52357

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

 JoVE Biology

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

1Department of Biology, University of Rochester, 2W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges


JoVE 52288

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.

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