JoVE General
Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels
1Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, 3Department of Anesthesiology, Medicine and Physiology, David Geffen School of Medicine, University of California, Los Angeles
We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.
JoVE General
Pull-down of Calmodulin-binding Proteins
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin
Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.
JoVE General
Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos
1Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, 2Departments of Neurology and Neurobiology, University of California, Los Angeles
Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.
JoVE General
Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye
Life Sciences Group, Hitachi High Technologies America
Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.
JoVE Immunology and Infection
Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding
Department of Pharmacology, School of Medicine, New York University
The crown region of different V3 loop sequences of the surface envelope glycoprotein (gp120) of HIV-1 can be structurally characterized in many cases by in silico folding of positions 10 to 22 of the loop using a state-of-the-art ab initio folding algorithm. Here we demonstrate the folding and evaluation of this region of the V3 loop from the R2 strain of HIV-1, a uniquely neutralization sensitive strain with puzzling functional properties.
JoVE Neuroscience
Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury
1Laboratory for NeuroRegeneration and Repair, Department of Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, 2Graduate School for Cellular and Molecular Neuroscience, University of Tuebingen
We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.
JoVE General
Identification of Protein Interacting Partners Using Tandem Affinity Purification
Section of Virology, Department of Medicine, Imperial College London
Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.
JoVE Bioengineering
Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound
1School of Biochemistry, University of Bristol, 2Smith and Nephew
This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.
JoVE Bioengineering
Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy
1Department of Computer Science and Engineering, Texas A&M University, 2Beckman Institute for Advanced Science and Technology, University of Illinois, 3Department of Electrical and Computer Engineering, Kettering University, 43Scan, 5Department of Veterinary Integrative Biosciences, Texas A&M University
The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.
JoVE General
Selective Capture of 5-hydroxymethylcytosine from Genomic DNA
1Department of Human Genetics, Emory University School of Medicine, 2Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago
Described is a two-step labeling process using β-glucosyltransferase (β-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.
JoVE General
Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.
1Program in Gene Function and Expression, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 2Broad Institute of Harvard and Massachusetts Institute of Technology, 3Division of Health Sciences and Technology, Massachusetts Institute of Technology, 4Program for Evolutionary Dynamics, Department of Organismic and Evolutionary Biology, Department of Mathematics, Harvard University, 5Department of Applied Mathematics, Harvard University, 6Department of Physics, Massachusetts Institute of Technology, 7Department of Systems Biology, Harvard Medical School, 8Department of Biology, Massachusetts Institute of Technology
The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.
JoVE General
RhoC GTPase Activation Assay
This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.
JoVE General
A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy
1Department of Neuroscience, Center for Neuroscience Research, Tufts School of Medicine, 2Department of Internal Medicine, Geriatrics & Gerontology, Wake Forest Baptist Medical Center, 3Department of Medicine, Boston University Medical Center
Mitochondrial fusion was measured by tracking the equilibration of photoconverted matrix-targeted GFP across the mitochondrial network over time. Thus far, only one cell could be subjected to an hour long kinetic analysis at a time. We present a method that simultaneously measures multiple cells, thereby speeding up the data collection process.
JoVE General
Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins
1Department of Radiation Oncology, Virginia Commonwealth University, 2Department of Biochemistry & Molecular Biology, Virginia Commonwealth University, 3Department of Anatomy & Neurobiology, Virginia Commonwealth University, 4Massey Cancer Center, Virginia Commonwealth University
This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.
JoVE General
Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans
1Department of Science, Borough of Manhattan Community College, City Universtiy of New York (CUNY), 2Department of Biology, Queens College, The City University of New York (CUNY), 3Biochemistry Program, The Graduate Center, Queens College, The City University of New York (CUNY)
We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-β signaling.
JoVE General
Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip
1Molecular Microbiology and Immunology, University of Missouri, 2Department of Surgery, University of Missouri, 3Child Health, University of Missouri
A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.
JoVE General
Methylated DNA Immunoprecipitation
1Department of Cancer Genetics and Developmental Biology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3These authors contributed equally., 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC, 5Photography/Video Production, Multi-Media Services, BC Cancer Agency, 6Department of Medical Genetics, Life Sciences Institute,, University of British Columbia - UBC
This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).
JoVE General
Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging
Division of Chemical Biology, New England Biolabs
SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.
JoVE Immunology and Infection
Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation
Center for Translational Systems Biology and Department of Neurology, Mount Sinai School of Medicine
We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.
JoVE General
IP-FCM: Immunoprecipitation Detected by Flow Cytometry
Department of Immunology, College of Medicine, Mayo Clinic
The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.
JoVE General
Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination
1Department of Biology, University of Washington, 2Howard Hughes Medical Institute, University of Washington, 3PRESTO, Japan Science and Technology Agency
We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.
JoVE General
In vivo and in vitro Studies of Adaptor-clathrin Interaction
Department of Biochemistry and Molecular Biology, Colorado State University
Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.
JoVE Immunology and Infection
A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay
1Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, 2Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, 3Department of Microbiology and Immunology, SUNY Upstate Medical University, 4Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center
The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.
JoVE General
Analyzing and Building Nucleic Acid Structures with 3DNA
1Department of Chemistry & Chemical Biology and BioMaPS Institute for Quantitative Biology, Rutgers - The State University of New Jersey, 2Department of Biological Sciences, Columbia University
The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.
JoVE Bioengineering
High-throughput Protein Expression Generator Using a Microfluidic Platform
We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.
JoVE General
Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software
Human Genetics, University of Michigan
The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.
JoVE General
Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth
1UCR Stem Cell Center, University of California, 2Department of Cell Biology and Neuroscience, University of California, 3Cell, Molecular, and Developmental Biology Graduate Program, University of California, 4Center for Research in Intelligent Systems, University of California
Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.
JoVE General
Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)
Stem Cell Research Unit, Medical University of Graz, Austria
This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.
JoVE Applied Physics
Echo Particle Image Velocimetry
Mechanical Engineering Department, University of New Hampshire
An echo particle image velocimetry (EPIV) system capable of acquiring two-dimensional fields of velocity in optically opaque fluids or through optically opaque geometries is described, and validation measurements in pipe flow are reported.
JoVE Neuroscience
Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat
1Helen Wills Neuroscience Institute, University of California Berkeley, 2Office of Laboratory Animal Care, University of California Berkeley, 3McGovern Institute for Brain Research & The Department of Brain and Cognitive Science, Massachusetts Institute of Technology, 4Integrative Biology Department, University of California Berkeley
Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.
JoVE Clinical and Translational Medicine
Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence
1Division of Pathology, University of Edinburgh, 2HistoRx Inc.
Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.
JoVE General
Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.
JoVE Chemistry
Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples
Department of Chemistry, University of Virginia
A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.
JoVE Neuroscience
Mechanical Manipulation of Neurons to Control Axonal Development
Department of Zoology, Michigan State University, East Lansing
Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.
JoVE General
Identification of protein complexes with quantitative proteomics in S. cerevisiae
1Department of Cellular and Physiological Sciences, University of British Columbia - UBC, 2Department of Biochemistry and Molecular Biology, University of British Columbia - UBC
Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.
JoVE General
Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro
Department of Biochemistry and Cell Biology, State University of New York
Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.
JoVE General
In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes
1Department of Biochemistry & Molecular Biology, Wayne State University School of Medicine, 2Cardiovascular Research Institute, Wayne State University School of Medicine, 3Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine
Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.
JoVE Clinical and Translational Medicine
Intranasal Administration of CNS Therapeutics to Awake Mice
A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.
JoVE Neuroscience
C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays
1Department of Molecular Biology, Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Lewis-Sigler Institute for Integrative Genomics, Princeton University
Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.
JoVE General
Preparation of Drosophila S2 cells for Light Microscopy
Department of Cell Biology and Anatomy, University of Arizona (UOA)
Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.
JoVE General
Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas
Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University
Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.
JoVE General
Using Laser Tweezers For Manipulating Isolated Neurons In Vitro
This video describes the manipulation of cultured neurons using laser tweezers in vitro.
JoVE General
In Ovo Electroporation in Embryonic Chick Retina
1Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 2Department of Biomedical Engineering, Rutgers University
The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.
JoVE Neuroscience
Preparation of Drosophila Central Neurons for in situ Patch Clamping
School of Life Sciences, Arizona State University
In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.
JoVE Neuroscience
Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants
1Department of Physical Medicine and Rehabilitation, VA Greater Los Angeles Healthcare System, 2Department of Pharmacology and Physiology, Institute for Neuroscience, The George Washington University School of Medicine and Health Sciences, 3Division of Genetics, Department of Medicine, Boston Children's Hospital, 4Howard Hughes Medical Institute, Boston Children's Hospital, 5Department of Pathology, Boston Children's Hospital, Harvard Medical School
The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.
JoVE Neuroscience
An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica
1Department of Biology, Case Western Reserve University, 2Department of Neurosciences, Case Western Reserve University, 3Department of Biomedical Engineering, Case Western Reserve University
We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.
JoVE Neuroscience
Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica
1Department of Biology, Case Western Reserve University, 2Department of Neurosciences, Case Western Reserve University, 3Department of Biomedical Engineering, Case Western Reserve University
In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
JoVE General
Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes
1Department of Molecular and Cellular Physiology, Stanford University, 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine
This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.
JoVE Clinical and Translational Medicine
Experimental Metastasis Assay
1Department of Biomedical Genetics, University of Rochester Medical Center, 2Department of Dermatology, University of Rochester Medical Center
This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.
JoVE Clinical and Translational Medicine
Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells
Laboratory for Orthopedic Research, Balgrist University Hospital, Zurich
The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.
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