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Immunology and Infection
Developmental Biology
 JoVE Biology

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

1Centre for Infectious Diseases and Microbiology, University of Sydney

JoVE 2781

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

 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 Biology

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

1Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)

JoVE 51582

Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis.

 JoVE Biology

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

1Bio-Rad Laboratories

JoVE 50948

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

 JoVE Biology

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

1Fraunhofer USA Center for Molecular Biotechnology

JoVE 51204

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

 JoVE Bioengineering

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

1Molekulare Mikrobiologie, Universität Osnabrück

JoVE 50810

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

 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 Immunology and Infection

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells

1Department of Pathology, New York University School of Medicine, 2Division of Infectious Diseases, Department of Medicine, Mount Sinai Medical Center, 3Division of Hematology and Oncology, Hess Center for Science and Medicine, Mount Sinai Medical Center

JoVE 51284

Dendritic cells (DCs) secrete IL-1β in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1β can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1β expression.

 JoVE Bioengineering

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

1Plate-Forme d'Imagerie Dynamique, Imagopole, Institut Pasteur, 2Department of Radiation Oncology, Stanford School of Medicine, 3Service Hospitalier Frédéric Joliot, Institut d'Imagerie Biomédicale, 4Vanderbilt School of Medicine, 5The Walter & Eliza Hall Institute of Medical Research, 6Unité INSERM U786, Institut Pasteur, 7Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur

JoVE 51549

Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.

 JoVE Medicine

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

1Center for Health Sciences, SRI International, 2Department of Chemistry and Biochemistry, University of California-Santa Cruz

JoVE 50960

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration. A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

 JoVE Biology

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

1Physical Biosciences Division, Lawrence Berkeley National Laboratory

JoVE 51715

This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.

 JoVE Immunology and Infection

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

1Lurie Family Imaging Center, Dana-Farber Cancer Institute, Harvard Medical School, 2Division of Pediatric Hematology/Oncology/Stem Cell Transplantation, Columbia University Medical Center

JoVE 50690

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

 JoVE Biology

Assaying Proteasomal Degradation in a Cell-free System in Plants

1Department of Biochemistry and Cell Biology, Stony Brook University, State University of New York

JoVE 51293

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular “tags” for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

 JoVE Immunology and Infection

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

1Animal and Plant Health Inspection Service, Wildlife Services, National Wildlife Research Center, USDA

JoVE 50853

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

 JoVE Biology

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

1Department of Biochemistry and Cell Biology, State University of New York

JoVE 2961

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 Application Notes

Profiling Changes in Receptor Tyrosine Kinase Phosphorylation using Antibody Arrays - ADVERTISEMENT

1Array Group, Assay Department, R&D Systems, Inc.

JoVE 4199

Proteome Profiler antibody arrays are a convenient and cost efficient way to screen for changes in receptor tyrosine kinase (RTK) phosphorylation without performing numerous immunoprecipitation (IP) Westerns. The ARY001 Human RTK array allows for the qualitative measurement of multiple RTKs in a single sample using chemiluminescence detection.

 JoVE Medicine

Analytical Techniques for Assaying Nitric Oxide Bioactivity

1Texas Therapeutics Institute, University of Texas Health Science Center at Houston, 2Deptartment of Pediatrics, Baylor College of Medicine

JoVE 3722

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

 JoVE Biology

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

1Department of Biotechnology, University of Verona, Verona, Italy, 2Department of Internal Medicine, University of Perugia, Perugia, Italy

JoVE 52459

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

 JoVE Neuroscience

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

1Department of Cellular and Physiological Sciences, Brain Research Centre, University of British Columbia

JoVE 50031

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

 JoVE Bioengineering

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

1Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, 2Division of Regenerative Medicine, Tulane National Primate Research Center, 3Department of Microbiology and Immunology, Tulane University School of Medicine, 4Department of Pharmacology, Tulane University School of Medicine

JoVE 50825

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

 JoVE Bioengineering

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

1Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University

JoVE 3612

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

 JoVE Biology

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

1Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University

JoVE 50588

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

 JoVE Immunology and Infection

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

1MRC Centre for Molecular Bacteriology and Infection, Division of Cell & Molecular Biology, Imperial College London, 2Preclinical Imaging, Caliper- A PerkinElmer Company

JoVE 50450

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with μCT imaging to create a 4D movie of C. rodentium infection.

 JoVE Application Notes

Simultaneous, Rapid, and Highly Efficient Protein Transfer Using the Trans-Blot Turbo Transfer System - ADVERTISEMENT

1Bio-Rad Laboratories

JoVE 3158

The Trans-Blot Turbo system reduces protein transfer protocols from gels to as little as 3 minutes, while maintaining high efficiency transfers and high throughput. The system enables protein transfer of 2 mini gels in 3 minutes and up to 4 mini gels in as little as 7 minutes.

 JoVE Immunology and Infection

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

1Department of Biological Sciences, University of Alabama

JoVE 50892

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

 JoVE Biology

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

1Lady Davis Institute for Medical Research, Jewish General Hospital, 2Department of Medicine, McGill University

JoVE 52230

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

 JoVE Bioengineering

Graphene Coatings for Biomedical Implants

1Department of Physics, Clemson University, 2Department of Pharmacology and Toxicology, East Carolina University, 3Department of Bioengineering, Clemson University, 4Center for Optical Materials Science and Engineering Technologies, Clemson University

JoVE 50276

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

 JoVE Biology

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

JoVE 4238

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.

 JoVE Immunology and Infection

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

1Department of Medicine, Vanderbilt University School of Medicine, 2Departments of Medicine and Immunology, Roswell Park Cancer Institute, 3Department of Medicine, University at Buffalo School of Medicine

JoVE 3925

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

 JoVE Biology

Isolation and Purification of Kinesin from Drosophila Embryos

1Department of Developmental and Cell Biology, School of Biosciences, University of California, Irvine

JoVE 3501

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 Medicine

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

1Department of Cell Biology, UT Southwestern Medical Center

JoVE 50369

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

 JoVE Immunology and Infection

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

1Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, 2First department of Internal Medicine, School of Medicine, Kyorin University, 3Neosilk Laboratory, Immuno Biological Laboratories Co., Ltd.

JoVE 2742

We designed a cell-free receptor binding assay in order to estimate the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to the receptors. It enables us to evaluate competitive inhibition of biotinylated GM-CSF binding to soluble GM-CSF receptor alpha by GM-CSF autoantibody with excellent reproducibility.

 JoVE Biology

Flow Cytometry-based Purification of S. cerevisiae Zygotes

1Department of Pathology, Case Western Reserve University School of Medicine, 2Cell Biology Program, Case Western Reserve University School of Medicine, 3Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine

JoVE 4197

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

 JoVE Biology

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

1Department of Biological Sciences and Purdue Center for Cancer Research, Purdue University

JoVE 1863

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 Neuroscience

SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

1Yerkes National Primate Research Center, Emory University, 2Department of Neurology, Institute of Clinical Medicine, Tsukuba University, 3Department of Pathology, New York University School of Medicine, 4Department of Neurology, Emory University

JoVE 1916

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the Aβ peptide. Using this protocol, we consistently detect monomeric and multimeric Aβ in cortical tissue from humans with Alzheimer's pathology.

 JoVE Biology

Immunoblot Analysis

1UVP, LLC, 2Proteomic Center, Keck Graduate Institute of Applied Life Sciences

JoVE 759

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 Application Notes

Advantages of the Trans-Blot® Turbo™ Transfer System Over Traditional Wet Tank and Semi-Dry Transfer Methods - ADVERTISEMENT

JoVE 3717

A demonstration of the advantages in speed, ease of use, and transfer efficiency of the Trans-Blot Turbo transfer system compared to conventional wet tank and semi-dry transfer methods.

 JoVE Application Notes

Western Blotting Troubleshooting Guide by Cell Signaling Technology - ADVERTISEMENT

1Cell Signaling Technology, Inc.

JoVE 5071

Here we provide an extensive western blot troubleshooting guide based on our many years of experience, offering solutions to save you valuable time and reagents.

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