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  JoVE Biology

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  JoVE Neuroscience

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  JoVE Immunology and Infection

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  JoVE Medicine

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  JoVE Bioengineering

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  JoVE Engineering

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  JoVE Chemistry

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  JoVE Behavior

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  JoVE Developmental Biology


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October, 2006
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 JoVE Biology

Preparation of Quality Inositol Pyrophosphates

1Medical Research Council (MRC), Cell Biology Unit and Laboratory for Molecular Cell Biology, University College London

JoVE 3027

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

 JoVE Immunology and Infection

Expansion of Human Peripheral Blood γδ T Cells using Zoledronate

1Department of Immunotherapeutics (Medinet), University of Tokyo Hospital, 2MEDINET Co., Ltd

JoVE 3182

A method to expand γδ T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived γδ T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of γδ T cells can be applied to autologous cellular immunotherapy of cancer.

 JoVE Biology

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

1Department of Neurology, Massachusetts General Hospital

JoVE 50282

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

 JoVE Chemistry

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

1Department of Chemistry and Biochemistry, University of Bern

JoVE 51385

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

 JoVE Biology

Pyrosequencing for Microbial Identification and Characterization

1Center for Biotechnology Education, Krieger School of Arts and Sciences, Johns Hopkins University, 2Qiagen Sciences, Inc.

JoVE 50405

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

 JoVE Biology

Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase

1Department of Genetics and Biochemistry, Clemson University

JoVE 3474

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

 JoVE Immunology and Infection

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

1Center for Molecular Bacteriology and Infection, Imperial College London

JoVE 50964

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

 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 Immunology and Infection

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

1Faculty of Medicine, Rappaport Institute, Technion - Israel Institute of Technology, 2The Pharmacy and Biochemistry Institute, Johannes Gutenberg University

JoVE 2390

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

 JoVE Bioengineering

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

1Departments of Chemistry and Materials Science and Engineering, University of Utah

JoVE 3949

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

 JoVE Biology

Method for the Isolation of Francisella tularensis Outer Membranes

1Department of Microbiology, University of Texas Southwestern Medical Center

JoVE 2044

A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.

 JoVE Immunology and Infection

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

1Department of Biology, San Diego State University, 2Computational Science Research Center, San Diego State University, 3Bioinformatics and Medical Informatics Research Center, San Diego State University, 4Department of Mathematics and Statistics, San Diego State University, 5Department of Computer Science, San Diego State University, 6Mathematics and Computer Science Division, Argonne National Laboratory, 7SPARC Committee, Broad Institute

JoVE 52854

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

 JoVE Biology

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

1Department of Pharmacy and Biotechnology, University of Bologna, 2CSGI, Department of Chemistry, University of Firenze

JoVE 52431

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

 JoVE Biology

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

1Department of Marine Sciences, University of Georgia (UGA)

JoVE 1086

We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.

 JoVE Chemistry

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

1Section of Molecular Genetics and Microbiology, The University of Texas at Austin, 2Department of Chemistry and Biochemistry, The University of Texas at Austin, 3The Institute of Cellular and Molecular Biology, The University of Texas at Austin

JoVE 50623

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

 JoVE Biology

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

1Institute of Toxicology and Genetics, Karlsruhe Institute of Technology - Campus North, 2Institute of Organic Chemistry, Karlsruhe Institute of Technology - Campus North, 3Institute of Organic Chemistry, Karlsruhe Institute of Technology - Campus South

JoVE 50439

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

 JoVE Medicine

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

1Department of Medicine, Northwestern University, 2Robert H. Lurie Cancer Center, Northwestern University, 3Center for Molecular Innovation and Drug Discovery, Northwestern University

JoVE 50873

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

 JoVE Biology

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

1Greenebaum Cancer Center, University of Maryland School of Medicine, 2Program in Molecular Medicine, University of Maryland School of Medicine, 3Department of Biochemistry & Molecular Biology, University of Maryland School of Medicine, 4Department of Pharmacology & Experimental Therapeutics, University of Maryland School of Medicine, 5Department of Pathology and Biochemistry & Molecular Biology, University of Maryland School of Medicine

JoVE 50512

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

 JoVE Medicine

Quantitative Analysis of Chromatin Proteomes in Disease

1Department of Anesthesiology, David Geffen School of Medicine at UCLA, 2Department of Medicine, David Geffen School of Medicine at UCLA, 3Department of Physiology, David Geffen School of Medicine at UCLA, 4Department of Internal Medicine, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah

JoVE 4294

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

 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 Medicine

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

1School of Biomedical Sciences and Pharmacy, Hunter Medical Research Institute, The University of Newcastle, 2School of Health and Human Sciences, Southern Cross University, 3School of Medicine and Public Health, The University of Newcastle

JoVE 51291

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury. This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

 Science Education: Essentials of Cell Biology

The ATP Bioluminescence Assay

JoVE Science Education

In fireflies, the luciferase enzyme converts a compound called luciferin into oxyluciferin, and produces light or “luminescence” as a result. This reaction requires energy derived from ATP in order to proceed, so researchers have exploited the luciferase-luciferin interaction to gauge ATP levels in cells. Given ATP’s role as the cell’s currency of energy, the ATP bioluminescence assay can provide insight into cellular metabolism and overall cell health.In this video, JoVE discusses cellular respiration, specifically reviewing how glucose metabolism results in ATP production. This is followed by principles behind the ATP bioluminescence assay and a generalized protocol for this technique. Finally, a survey of how researchers are currently using the ATP bioluminescence assay to evaluate cell viability in a variety of experimental conditions.

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