1Medical Research Council (MRC), Cell Biology Unit and Laboratory for Molecular Cell Biology, University College London
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.
Published September 3, 2011. Keywords: Molecular Biology, Polyacrilamyde Gel Electrophoresis (PAGE), inositol hexakisphosphate (IP6), phytic acid, diphosphoinositol pentakisphosphate (IP7), bisdiphoshoinositol tetrakisphosphate (IP8), IP6-kinase (IP6K), PP-IP5K, VIP1
JoVE Immunology and Infection
1Department of Immunotherapeutics (Medinet), University of Tokyo Hospital, 2MEDINET Co., Ltd
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.
Published September 9, 2011. Keywords: Immunology, γδ T Cell, zoledronate, PBMC, peripheral blood mononuclear cells
1Department of Neurology, Massachusetts General Hospital
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.
Published June 1, 2014. Keywords: Cellular Biology, Luciferases, Gene Transfer Techniques, Transfection, High-Throughput Screening Assays, Transfections, Robotics
1Department of Chemistry and Biochemistry, University of Bern
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.
Published April 3, 2014. Keywords: Chemistry, Nucleic acid analogues, Bioorganic Chemistry, PCR, primer extension reactions, organic synthesis, PAGE, HPLC, nucleoside triphosphates
1Center for Biotechnology Education, Krieger School of Arts and Sciences, Johns Hopkins University, 2Qiagen Sciences, Inc.
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.
Published August 22, 2013. Keywords: Microbiology, Genetics, Molecular Biology, Basic Protocols, Genomics, Eukaryota, Bacteria, Viruses, Bacterial Infections and Mycoses, Virus Diseases, Diagnosis, Therapeutics, Equipment and Supplies, Technology, Industry, and Agriculture, Life Sciences (General), Pyrosequencing, DNA, Microbe, PCR, primers, Next-Generation, high-throughput, sequencing
1Department of Genetics and Biochemistry, Clemson University
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.
Published December 19, 2011. Keywords: Molecular Biology, Acetate kinase, acetate, acetyl phosphate, pyrophosphate, PPi, ATP
JoVE Immunology and Infection
1Center for Molecular Bacteriology and Infection, Imperial College London
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.
Published November 22, 2013. Keywords: Infection, Bacterial Infections, Infection, Disease Models, Animal, Bacterial Infections and Mycoses, Galleria mellonella, Legionella pneumophila, insect model, bacterial infection, Legionnaires' disease, haemocytes
1Department of Pharmacology and Toxicology, University of Toronto
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.
Published September 3, 2014. Keywords: Neurobiology, brain, synapse, western blot, ultracentrifugation, SPM, PSD
JoVE Immunology and Infection
1Faculty of Medicine, Rappaport Institute, Technion - Israel Institute of Technology, 2The Pharmacy and Biochemistry Institute, Johannes Gutenberg University
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.
Published October 19, 2010. Keywords: Immunology, tRNA, methylation, DNA methyltransferase 2, Entamoeba histolytica
1Departments of Chemistry and Materials Science and Engineering, University of Utah
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.
Published July 11, 2012. Keywords: Bioengineering, Materials Science, Chemical Engineering, enzyme immobilization, polymer modification, Nafion, enzyme stabilization, enzyme activity assays
1Department of Microbiology, University of Texas Southwestern Medical Center
A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.
Published June 29, 2010. Keywords: Microbiology, Francisella, tularemia, outer membrane protein, sucrose density gradient centrifugation, membrane isolation, osmotic lysis, spheroplast
1Department of Pharmacy and Biotechnology, University of Bologna, 2CSGI, Department of Chemistry, University of Firenze
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.
Published February 5, 2015. Keywords: Biochemistry, Escherichia coli, protein co-expression, compatible plasmids, complementation test, DNA polymerase III, mutator strains
1Department of Marine Sciences, University of Georgia (UGA)
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.
Published February 18, 2009. Keywords: Microbiology, transcriptomics, bacterioplankton, mRNA, microbial communities, gene expression
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
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.
Published September 16, 2013. Keywords: Chemistry, Membrane Lipids, Toll-Like Receptors, Endotoxins, Glycolipids, Lipopolysaccharides, Lipid A, Microbiology, Lipids, lipid A, Bligh-Dyer, thin layer chromatography (TLC), lipopolysaccharide, mass spectrometry, Collision Induced Dissociation (CID), Photodissociation (PD)
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
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.
Published September 10, 2013. Keywords: Developmental Biology, Biochemistry, Vertebrates, Zebrafish, environmental effects (biological and animal), genetics (animal), life sciences, animal biology, animal models, biochemistry, bioengineering (general), Hormones, Hormone Substitutes, and Hormone Antagonists, zebrafish, Danio rerio, chemical screening, luciferase, glucocorticoid, stress, high-throughput screening, receiver operating characteristic curve, in vivo, animal model
1Department of Medicine, Northwestern University, 2Robert H. Lurie Cancer Center, Northwestern University, 3Center for Molecular Innovation and Drug Discovery, Northwestern University
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.
Published September 18, 2013. Keywords: Medicine, Urogenital System, Male Urogenital Diseases, Surgical Procedures, Operative, Life Sciences (General), Prostate Cancer, Metastasis, Mouse Model, Drug Discovery, Molecular Biology
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
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.
Published August 31, 2013. Keywords: Cellular Biology, Transcription Factors, Vitamin D, Drug Discovery, Enzyme-Linked Immunosorbent Assay (ELISA), DNA-binding, transcription factor, drug screening, antibody
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
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.
Published December 28, 2012. Keywords: Medicine, Molecular Biology, Immunology, Genetics, Genomics, Physiology, Protein, DNA, Chromatin, cardiovascular disease, proteomics, mass spectrometry
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
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.
Published February 4, 2014. Keywords: Medicine, Brain slices, in vitro, excitotoxicity, brain injury, Mature brain tissue, Stimulation, stroke