JoVE Application Notes
Analytical HPLC to Preparative HPLC: Scale Up Techniques using a Natural Product Extract - ADVERTISEMENT
Pharmaceutical Business Operations, Waters Corporation
Using the Waters AutoPurification™ System, separation methods can be developed on an analytical scale and transferred to preparatory scale on the same system.
JoVE General
Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University
An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.
JoVE Application Notes
Basic Principles for Purification Using Supercritical Fluid Chromatography - ADVERTISEMENT
Chemistry Commercial Operations, Waters Corporation
Basic principles for the separation of compounds from mixtures using supercritical fluid chromatography (SFC) are similar to the fundamentals of preparative liquid chromatography.
JoVE General
A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples
1Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco - UCSF, 2Buck Institute for Age Research, 3Department of Chemistry, Purdue University
Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.
JoVE General
Isolation of Immune Cells from Primary Tumors
1Tumor Immunity and Tolerance Section, Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, National Cancer Institute - Frederick, 2KEWB Productions
In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.
JoVE Immunology and Infection
Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography
1Department of Microbial and Environmental Genomics, The J. Craig Venter Institute, 2Department of Synthetic Biology and Bioenergy, The J. Craig Venter Institute
We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.
JoVE General
Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAΔ41
MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamAΔ41) from M. magneticum AMB-1.
JoVE General
High-Throughput Measurement and Classification of Organic P in Environmental Samples
School of Engineering, University of Vermont
This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.
JoVE Immunology and Infection
Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means
1Centro de Biología Celular y Molecular de Enfermedades, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP), 2Acharya Nagarjuna University, 3Departamento de Medios y Creativo, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP)
The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.
JoVE General
Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein
Department of Biological Sciences, University of Illinois Chicago - UIC
In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.
JoVE Clinical and Translational Medicine
Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma
1Institute of Pathology, Laboratory of Molecular Tumor Pathology, Charité - Universitätsmedizin Berlin, 2Institute for Chemistry and Biochemistry, Free University Berlin, 3Laboratory for Functional Genomics Charité (LFGC), Charité - Universitätsmedizin Berlin, 4Comprehensive Cancer Center Charité, Charité - Universitätsmedizin Berlin
This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).
JoVE Neuroscience
Double Fluorescence in situ Hybridization in Fresh Brain Sections
1Department of Brain and Cognitive Sciences, University of Rochester, 2Center for Visual Science, University of Rochester
This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.
JoVE General
Microarray Analysis for Saccharomyces cerevisiae
Vermont Genetics Network, The University of Vermont
In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.
JoVE General
Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae
Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.
JoVE General
An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase
Department of Biochemistry, University of Toronto
The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.
JoVE Immunology and Infection
Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells
This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.
JoVE Immunology and Infection
Facilitating the Analysis of Immunological Data with Visual Analytic Techniques
1Department of Paediatrics, Division of Infectious and Immunological Diseases, Child and Family Research Institute, University of British Columbia, 2Department of Computer Science, University of British Columbia, 3Department of Psychology, University of British Columbia
Visual analytics (VA) is a new approach of analyzing data interactively. In this video, we discuss the data overload problem brought on by high-throughput biological experiments, and propose VA as a solution to such problem. The video demonstrates analysis within and between immunological datasets using a VA tool called Tableau.
JoVE Immunology and Infection
A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms
1Department of Biology, University of Texas San Antonio - UTSA, 2South Texas Center for Emerging Infectious Diseases, University of Texas San Antonio - UTSA
We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.
JoVE General
Thin Sectioning of Slice Preparations for Immunohistochemistry
1Department of Medicine, Yonsei University College of Medicine, Severance Hospital, 2Department of Psychiatry, Harvard Medical School
The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.
JoVE General
Chromatographic Purification of Highly Active Yeast Ribosomes
1Department of Cell Biology and Molecular Genetics, University of Maryland, 2Department of Biotechnology and Microbiology, Vilnius University
Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.
JoVE Immunology and Infection
Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes
Department of Internal Medicine B, University Medicine Greifswald
The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.
JoVE General
Single Cell Fate Mapping in Zebrafish
1Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 2Division of Hematology/Oncology, Cincinnati Children's Hospital Medical Center
A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.
JoVE Immunology and Infection
Recombinant Retroviral Production and Infection of B Cells
1Department of Microbiology and Immunology, Columbia University College of Physicians and Surgeons, 2Herbert Irving Comprehensive Cancer Center, Columbia University
An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.
JoVE General
Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
School of Biotechnology, Department of Proteomics, Royal Institute of Technology
A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.
JoVE General
Bacterial Gene Expression Analysis Using Microarrays
Department of Environmental Toxicology, University of California Santa Cruz - UCSC
JoVE General
The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis
1The Warren Alpert Medical School of Brown University, 2Laboratorio de Investigacion de Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, 3Department of International Health, Johns Hopkins Bloomberg School of Public Health, 4Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College London
The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.
JoVE General
A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica
1Department of Biological Sciences, The University of Alabama, Huntsville, 2USDA-APHIS-PPQ, Center for Plant Health Science and Technology
We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).
JoVE Immunology and Infection
Production and Titering of Recombinant Adeno-associated Viral Vectors
1School of Medical Sciences, College of Life Sciences and Medicine, University of Aberdeen, 2Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, University of New South Wales, 3Department of Biochemistry and Molecular Biophysics, Columbia University
Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.
JoVE Immunology and Infection
Mass Spectrometric Analysis of Glycosphingolipid Antigens
1Undergraduate Program, Rice University, 2Proteomics Facility, Department of Pathology, University of Texas MD Anderson Cancer Center, 3Department of Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center, 4University of Texas Graduate School of Biological Sciences at Houston
A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.
JoVE General
Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)
JoVE General
Spinal Cord Electrophysiology
1The Salk Institute for Biological Studies, Howard Hughes Medical Institute and Gene Expression Laboratory, 2Biology Graduate Program, University of California San Diego - UCSD
A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.
JoVE General
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
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 General
Processing the Loblolly Pine PtGen2 cDNA Microarray
1Warnell School of Forestry and Natural Resources, University of Georgia (UGA), 2Instituto de Biologia Experimental e Tecnológica, Instituto Tecnologia Química e Biológica UNL, Av. da República
The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.
JoVE Immunology and Infection
Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays
Plant Pathology and Microbiology, Texas A&M University
The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.
JoVE General
Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors
1Institute for Research in Biomedicine, Bellinzona (Switzerland), 2Dipartimento di Biologia e Genetica per le Scienze Mediche, Universitá degli Studi di Milano
A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.
JoVE General
Isolation of CD133+ Liver Stem Cells for Clonal Expansion
1Department of Pediatrics and Pharmacology, Pennsylvania State College of Medicine, 2Department of Pharmacology, Pennsylvania State College of Medicine, 3Department of Pediatrics, University of California Los Angeles, School of Medicine
Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.
JoVE Clinical and Translational Medicine
Dorsal Column Steerability with Dual Parallel Leads using Dedicated Power Sources: A Computational Model
Boston Scientific , Neuromodulation
Using a mathematical model of spinal cord stimulation, we found that a multi-source system with independent power sources for each contact can target more central points of stimulation on the dorsal column (100 vs 3) and has 50-fold more field steering resolution (0.02mm vs 1mm) than a single-source system.
JoVE Immunology and Infection
Isolation of Precursor B-cell Subsets from Umbilical Cord Blood
1Department of Pathology and Anatomical Sciences, University of Missouri-Columbia, 2Laboratory for Infectious Disease Research, University of Missouri-Columbia
Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.
JoVE General
Purification of Hsp104, a Protein Disaggregase
Department of Biochemistry and Biophysics, University of Pennsylvania
Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.
JoVE General
Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran
1Department of Biological Sciences, Vanderbilt University, 2Department of Chemical and Systems Biology, Stanford University
This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.
JoVE General
Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos
Department of Cell Biology, University of Massachusetts Medical School
We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.
JoVE General
Examining BCL-2 Family Function with Large Unilamellar Vesicles
Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented.
JoVE Clinical and Translational Medicine
Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients
Our experiment will show how to perform a sequencing analysis of bacterial species translocating in peripheral blood of HIV positive patients.
JoVE Chemistry
Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies
1Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, 2The University of Melbourne
Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.
JoVE Neuroscience
Selection of Aptamers for Amyloid β-Protein, the Causative Agent of Alzheimer's Disease
1Department of Neurology, David Geffen School of Medicine, 2Molecular Biology Institute, University of California, Los Angeles, 3Brain Research Institute, University of California, Los Angeles
Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease.
JoVE General
Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform
School of Health Sciences, Purdue University
Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.
JoVE Immunology and Infection
Visualizing Dengue Virus through Alexa Fluor Labeling
1Defence Medical and Environmental Research Institute, DSO National Laboratories, 2Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 3Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School
Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.
JoVE Neuroscience
Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex
1Neuroinformatics DTC, University of Edinburgh, 2Centre for Integrative Physiology, University of Edinburgh
We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.
JoVE General
Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization
1Department of Cancer Genetics, BC Cancer Research Centre, 2Deeley Research Centre, BC Cancer Agency, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency
This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.
JoVE General
In vitro Reconstitution of the Active T. castaneum Telomerase
Gene Expression and Regulation, The Wistar Institute, University of Pennsylvania
Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
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