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  JoVE Neuroscience

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  JoVE Medicine

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  JoVE Developmental Biology


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 JoVE Biology

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

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

JoVE 52099

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.

 JoVE Biology

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

1Bio-Rad Laboratories

JoVE 50948

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

 JoVE Application Notes

Western Blotting Troubleshooting Guide by Cell Signaling Technology - ADVERTISEMENT

1Cell Signaling Technology, Inc.

JoVE 5071

Here we provide an extensive western blot troubleshooting guide based on our many years of experience, offering solutions to save you valuable time and reagents.

 JoVE Application Notes

Western Blotting Protocol - ADVERTISEMENT

1Cell Signaling Technology, Inc.

JoVE 5072

Here we provide a comprehensive western blot protocol developed and used at Cell Signaling Technology that includes optimal reagents and support information to ensure optimal results.

 JoVE Biology

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

1Department of Nephrology, Children's Hospital of Pittsburgh of UPMC, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine

JoVE 50867

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

 JoVE Biology

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

1Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven and Leuven Institute for Neuroscience and Disease (LIND)

JoVE 50523

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

 JoVE Neuroscience

Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

1Department of Molecular Physiology and Biophysics, Baylor College of Medicine, 2Department of Neuroscience, Baylor College of Medicine, 3Department of Neuroscience, University of California at San Diego, 4National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine

JoVE 50783

To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.

 JoVE Biology

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

1Diller Cancer Research Building, University of California, San Francisco

JoVE 51149

This protocol explores the latest advancements in performing Western blot analyses. These novel modifications employ a Bis-Tris gel system with a 35 min electrophoresis run time, a 7 min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

 JoVE Biology

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

1Département de Génomique Fonctionnelle et Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 2Center for Life NanoScience, Istituto Italiano di Tecnologia

JoVE 51814

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

 JoVE Biology

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

1Fraunhofer USA Center for Molecular Biotechnology

JoVE 51204

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

 JoVE Neuroscience

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

1Department of Cellular and Physiological Sciences, Brain Research Centre, University of British Columbia

JoVE 50031

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

 JoVE Biology

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

1Department of Biology, Ball State University, 2Division of Nephrology, Cincinnati Children's Hospital

JoVE 52428

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

 JoVE Application Notes

Simultaneous, Rapid, and Highly Efficient Protein Transfer Using the Trans-Blot Turbo Transfer System - ADVERTISEMENT

1Bio-Rad Laboratories

JoVE 3158

The Trans-Blot Turbo system reduces protein transfer protocols from gels to as little as 3 minutes, while maintaining high efficiency transfers and high throughput. The system enables protein transfer of 2 mini gels in 3 minutes and up to 4 mini gels in as little as 7 minutes.

 JoVE Biology

Induction and Analysis of Epithelial to Mesenchymal Transition

1Antibody Development Department, R&D Systems, Inc., 2Stem Cells and Developmental Biology Department, R&D Systems, Inc.

JoVE 50478

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

 JoVE Bioengineering

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

1Department of Chemical and Biomolecular Engineering, Ohio University, 2Biomedical Engineering Program, Russ College of Engineering and Technology, Ohio University, 3Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School

JoVE 51023

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins. Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

 JoVE Chemistry

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

1Department of Chemistry, The Scripps Research Institute, 2Worm Institute of Research and Medicine (WIRM), The Scripps Research Institute

JoVE 50908

The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity.

 JoVE Environment

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

1Institute for Molecular Biotechnology, Bio7, RWTH Aachen University, 2Fraunhofer Institute for Molecular Biology and Applied Ecology

JoVE 51711

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

 JoVE Biology

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

1Stowers Institute for Medical Research, 2Department of Biochemistry & Molecular Biology, Kansas University Medical Center

JoVE 51720

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.

 JoVE Biology

Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding

1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 3School of Medicine, University of Washington

JoVE 3059

An in vitro method for preparing functional glucocorticoid receptor (GR)•hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

 JoVE Bioengineering

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

1Center for Applied Proteomics and Molecular Medicine, George Mason University, 2Ceres Nanosciences

JoVE 51789

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

 JoVE Immunology and Infection

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

1Inflammation Research Center, VIB, 2Department of Biomedical Molecular Biology, Ghent University

JoVE 52871

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

 JoVE Immunology and Infection

A Protocol for Analyzing Hepatitis C Virus Replication

1Liver Program at Regenerative Medicine Institute, Department of Biomedical Sciences, Department of Surgery, Cedars-Sinai Medical Center, 2Department of Surgery, David Geffen School of Medicine at UCLA

JoVE 51362

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

 JoVE Chemistry

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

1Nanomedicine Research Center, Department of Neurosurgery, Cedars-Sinai Medical Center

JoVE 50668

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

 JoVE Bioengineering

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

1School of Materials Science and Engineering, Nanyang Technological University

JoVE 52845

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

 JoVE Biology

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

1Department of Biological, Geological, and Environmental Sciences, Cleveland State University

JoVE 52772

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

 JoVE Biology

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

1Institute of Biomembranes and Bioenergetics, National Research Council

JoVE 52849

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

 JoVE Bioengineering

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

1Polymer and Composite Engineering (PaCE) Group, Institute of Materials Chemistry and Research, University of Vienna, 2Department of Chemical Engineering, University College London, 3Polymer and Composite Engineering (PaCE) Group, Department of Chemical Engineering, Imperial College London

JoVE 51432

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

 JoVE Immunology and Infection

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

1Institute for Virology, Philipps-University Marburg

JoVE 51415

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

 JoVE Biology

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

1Integrated Science Center, Biology Department, The College of William & Mary

JoVE 50921

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

 JoVE Biology

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

1Department of Molecular Genetics, The Ohio State University

JoVE 52030

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein’s fluorescence intensity to that of an appropriate internal standard.

 JoVE Neuroscience

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

1Department of Pharmacology and Toxicology, University of Toronto

JoVE 51896

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

 JoVE Biology

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

1Department of Anatomy and Cell Biology, University of Iowa Carver College of Medicine

JoVE 50493

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

 JoVE Immunology and Infection

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

1Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health canada, 2National Institute for the Control of Pharmaceutical and Biological Products, The State Food and Drug Administration, Beijing, 3Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 4Microbiology Department, Faculty of Medicine, King Abdulaziz University, 5National Microbiology Laboratory, Public Health Agency of Canada

JoVE 2784

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

 JoVE Biology

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

1Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford

JoVE 51206

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

 JoVE Medicine

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

1Department of Cardiology, University of Heidelberg, 2Department of Vascular Surgery, University of Heidelberg, 3Department of Cardiology, SLK Hospital am Plattenwald

JoVE 50542

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

 JoVE Biology

Assaying Proteasomal Degradation in a Cell-free System in Plants

1Department of Biochemistry and Cell Biology, Stony Brook University, State University of New York

JoVE 51293

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular “tags” for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

 JoVE Biology

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

1Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital, 2Department of Surgery, University of Toronto, 3Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill

JoVE 3932

The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.

 JoVE Neuroscience

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

1RG Neuroplasticity, Leibniz Institute for Neurobiology, 2Department of Cell Biology, Utrecht University

JoVE 51310

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

 JoVE Biology

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

1Institute of Chemistry, Technical University of Berlin, 2The Vollum Institute, Oregon Health & Science University

JoVE 50201

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

 JoVE Medicine

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

1Department of Pathology, National Prion Disease Pathology Surveillance Center, Case Western Reserve University School of Medicine, 2Department of Neurology, National Prion Disease Pathology Surveillance Center, Case Western Reserve University School of Medicine

JoVE 3788

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

 JoVE Biology

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

1Lady Davis Institute and Department of Oncology, McGill University, 2Department of Oncology-Pathology, Karolinska Institutet, 3Goodman Cancer Centre and Department of Biochemistry, McGill University

JoVE 51455

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

 JoVE Bioengineering

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

1Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University

JoVE 3612

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

 JoVE Immunology and Infection

Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries

1The Recombinant Antibody Network, 2The Banting and Best Department of Medical Research, University of Toronto, 3Antibiome Center, University of California, San Francisco at Mission Bay, 4Department of Biochemistry and Molecular Biology, The University of Chicago

JoVE 51492

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

 JoVE Biology

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

1Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 2Deparment of Biochemistry, Research and Innovation Centre, University of Regina, 3Department of Medical Genetics and Microbiology, University of Toronto

JoVE 4057

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

 JoVE Developmental Biology

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

1Department of Anatomy and Neuroscience, The University of Melbourne, 2The Florey Institute of Neuroscience and Mental Health Research, The University of Melbourne

JoVE 52179

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

 JoVE Bioengineering

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

1Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, 2Department of Chemical and Biomolecular Engineering, University of Tennessee

JoVE 50981

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

 JoVE Immunology and Infection

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

1Cluster of Excellence CellNetworks, Department of Infectious Diseases, Virology, Heidelberg University, 2Department of Infectious Diseases, Virology, Heidelberg University

JoVE 3819

We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.

 JoVE Immunology and Infection

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

1Department of Animal Science, Texas A&M University, 2Department of Biology, Texas A&M University, 3Department of Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University

JoVE 50323

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

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