Molecular Biology:
A discipline concerned with studying biological phenomena in terms of the chemical and physical interactions of molecules.
JoVE General
Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay
Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine
We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase.
JoVE General
Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity
DOE Plant Research Laboratory, Michigan State University
A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.
JoVE General
Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit
Department of Developmental and Cell Biology, University of California, Irvine (UCI)
The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.
JoVE General
Electrophoretic Separation of Proteins
Proteomic Center, Keck Graduate Institute of Applied Life Sciences
In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).
JoVE General
Immunoblot Analysis
1UVP, LLC, 2Proteomic Center, Keck Graduate Institute of Applied Life Sciences
Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.
JoVE General
Staining Proteins in Gels
1Keck Graduate Institute of Applied Life Sciences, UVP, LLC, 2Proteomic Center, Keck Graduate Institute of Applied Life Sciences
Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.
JoVE General
Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System
1Department of Biology, University of Western Ontario, 2Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada
We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.
JoVE General
MISSION esiRNA for RNAi Screening in Mammalian Cells
Max Planck Institute of Molecular Cell Biology and Genetics
Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.
JoVE General
Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis
Department of Ophthalmology, SUNY Upstate Medical University
Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.
JoVE General
A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract
Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.
JoVE General
DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses
1Department of Integrative Oncology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency, 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC
This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.
JoVE General
Dissection of Saccharomyces Cerevisiae Asci
Department of Biology, Concordia University
Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.
JoVE General
An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species
Warnell School of Forestry and Natural Resources, University of Georgia (UGA)
Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.
JoVE General
Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer
Shimadzu, Scientific Instruments
This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.
JoVE Neuroscience
Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology
National Eye Institute, National Institutes of Health
We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.
JoVE General
Isolation and Purification of Kinesin from Drosophila Embryos
This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.
JoVE General
Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes
Department of Environmental Science and Engineering, California Institute of Technology - Caltech
This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.
JoVE General
Isolation and Derivation of Mouse Embryonic Germinal Cells
The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.
JoVE General
Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)
1School of Biological Sciences, Nanyang Technological University, Singapore - NTU, 2Singapore-MIT Alliance for Reserach and Technology (SMART)
Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.
JoVE Clinical and Translational Medicine
Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock
Department of Surgery, University of Pittsburgh
The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.
JoVE Clinical and Translational Medicine
Pseudofracture: An Acute Peripheral Tissue Trauma Model
1Department of Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Aachen Medical Center
Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.
JoVE Immunology and Infection
A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors
1Department of Entomology, Texas A&M University (TAMU), 2Department of Molecular and Cellular Medicine, Texas A&M University (TAMU)
This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.
JoVE Immunology and Infection
Following Cell-fate in E. coli After Infection by Phage Lambda
1Department of Physics, University of Illinois at Urbana-Champaign, 2Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, 3Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine
This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.
JoVE Bioengineering
Quantitative FRET (Förster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination
Department of Bioengineering, Bourns College of Engineering, University of California, Riverside
A novel method involving quantitative analysis of FRET (Förster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.
JoVE General
Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast
Department of Biological Sciences and Purdue Center for Cancer Research, Purdue University
Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.
JoVE Editorial
October 2011: This Month in JoVE
Here are some highlights from the October 2011 Issue of Journal of Visualized Experiments (JoVE).
JoVE Editorial
December 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here are some highlights from the December 2012 Issue of Journal of Visualized Experiments (JoVE).
JoVE Clinical and Translational Medicine
Dissection of Human Vitreous Body Elements for Proteomic Analysis
Department of Ophthalmology and Visual Sciences, Omics Laboratory, University of Iowa
This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.
JoVE General
A high-throughput method to globally study the organelle morphology in S. cerevisiae
Department of Cellular and Physiological Sciences, University of British Columbia - UBC
GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.
JoVE Bioengineering
Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study
Department of Engineering Mechanics, University of Nebraska-Lincoln
A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate the molecular/cellular mechanisms of blast-induced traumatic brain injury.
JoVE Editorial
May 2011: This Month in JoVE
The main highlights for our May issue include methods for measuring cognition in zero gravity, isolating mosquito immune cells, engineering recombinant SARS vaccines, and detecting tumors with thermal imaging. In addition, procedures for isolating neural stem cells from human fetal brain and culturing antigen-presenting liver cells will also be released.
JoVE General
The ITS2 Database
1Department of Bioinformatics, Biocenter, University of Würzburg, 2Institute of Pharmacy and Food Chemistry, University of Würzburg
The ITS2 Database is a workbench for phylogenetic inference simultaneously considering sequence and secondary structure of the internal transcribed spacer 2. This includes data collection with accurate annotation, structure prediction, multiple sequence-structure alignment and fast tree calculation. In a nutshell, this workbench simplifies first phylogenetic analyses to a few clicks.
JoVE General
Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions
Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute
AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.
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 Immunology and Infection
An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium
Department of Population Health, University of Georgia
We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.
JoVE Editorial
June 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Back in 1905, in what is now the Czech Republic, Eduard Zirm performed the first corneal transplantation surgery (keratoplasty), which restored vision to a patient blinded by corneal injury. Today, eye banks all over the world prepare, store, and distribute donated corneas to hospitals so that thousands of sight-saving keratoplasties can be performed every year. In June 2012, JoVE has its eye on two research groups, one from Italy and the other from Michigan, who demonstrate two distinct methods for corneal graft preparation prior to transplantation.
JoVE Immunology and Infection
Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy
1Institute of Plant Sciences, University of Graz, 2Institute for Electron Microscopy and Fine Structure Research, Graz University of Technology
This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.
JoVE Immunology and Infection
Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus
1Center for Autophagy Research, Department of Internal Medicine, UT Southwestern Medical Center, 2Department of Microbiology, UT Southwestern Medical Center
We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (γHV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for γHV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.
JoVE General
Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology
1Department of Biology, Syracuse University, 2Department of Science Teaching, Syracuse University
Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.
JoVE Editorial
May 2012: This Month in JoVE
Here are some highlights from the May 2012 Issue of Journal of Visualized Experiments (JoVE).
JoVE Editorial
September 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
This September in JoVE, researchers from the School of Medicine at the Free University of Berlin demonstrate a novel method for studying how stroke patients compensate for visual field defects. To do this, our authors make use of a driving simulator complete with brakes, a steering wheel, and turn signals. Using driving simulation software and sophisticated eye tracking, researchers can compare the gaze behavior of stroke patients as they navigate through virtual driving courses with varying degrees of complexity. Though posterior cerebral artery infarction can lead to similar visual deficits in patients, some are able to navigate through the driving courses by developing compensatory eye movements, while others crash into dangerous obstacles, like wild boars. Through the analysis of compensatory gaze behavior employed by patients, our authors see great potential for using driving simulation as a tool to rehabilitate stroke patients trying to overcome the blind spots in their visual fields.
JoVE General
Genotyping of Plant and Animal Samples without Prior DNA Purification
Thermo Scientific Molecular Biology Products, Thermo Fisher Scientific
The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.
JoVE General
Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays
Department of Biochemistry and Molecular Biology, University of Chicago
We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.
JoVE Clinical and Translational Medicine
A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study
The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.
JoVE Clinical and Translational Medicine
Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats
Medicine/Nephrology, Indiana University School of Medicine
A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.
JoVE Clinical and Translational Medicine
Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens
1Molecular Diabetology, Paul Langerhans Institute Dresden, 2Department of GI-, Thoracic- and Vascular Surgery, University Hospital Carl Gustav Carus, University of Technology Dresden, 3Department of Endocrinology and Metabolism, Metabolic Unit University of Pisa, 4Labs DC0522, Lilly Corporate Center, 5Genomics, Faculty of Medicine Imperial College London, 6Vital-IT, SIB Swiss Institute of Bioinformatics, 7Clinical Biochemistry, Hannover Medical School, 8Cell Physiology and Metabolism, Medical School, University of Geneva, 9Department of Pathology, University Hospital Carl Gustav Carus, University of Technology Dresden, 10R&D DIAB Division / Translational Medicine, Sanofi-Aventis
Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.
JoVE General
Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit
Department of Physiology and Biophysics, University of California, Irvine (UCI)
JoVE Clinical and Translational Medicine
Technique to Collect Fungiform (Taste) Papillae from Human Tongue
1Department of Basic Science and Craniofacial Biology, College of Dentistry, New York University, 2Department of Internal Medicine and Department of Psychiatry, School of Medicine, Washington University in St. Louis, 3Veterans Affairs Medical Center, 4School of Dental Medicine, Department of Biochemistry, University of Pennsylvania-School of Medicine, 5 , Monell Chemical Senses Center, 6Monell Chemical Senses Center
Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.
JoVE General
Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms
Department of Chemistry, McGill University
ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.
JoVE General
Glycan Profiling of Plant Cell Wall Polymers using Microarrays
1Australian Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, 2Plant Cell Biology Research Centre, School of Botany, University of Melbourne, 3CSIRO Plant Industry, Black Mountain Laboratories, 4Department of Plant Biology and Biotechnology, University of Copenhagen
A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.
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