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JoVE Science Education

General Laboratory Techniques

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Basic Methods in Cellular and Molecular Biology

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Model Organisms I

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 JoVE Biology

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

1Department of Pharmacology, University of Illinois at Chicago


JoVE 50529

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

 JoVE Biology

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

1Division of Virology, Department of Pathology, University of Cambridge


JoVE 51656

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

 JoVE Biology

Identifying Protein-protein Interaction Sites Using Peptide Arrays

1Institute of Chemistry, The Hebrew University of Jerusalem


JoVE 52097

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

 JoVE Biology

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

1Neuroscience Center of Excellence, Louisiana State University Health Sciences Center


JoVE 50968

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

 JoVE Biology

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

1Department of Pharmaceutical Biotechnology and Molecular Biology, Centre for Pharmaceutical Research, Vrije Universiteit Brussel


JoVE 3937

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

 JoVE Biology

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

1Dept. Of Cell Biology and Molecular Genetics, University of Maryland


JoVE 1963

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

 JoVE Biology

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

1Language and Genetics Department, Max Planck Institute for Psycholinguistics, 2Donders Institute for Brain, Cognition and Behaviour


JoVE 51438

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

 JoVE Biology

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

1Department of Biochemistry, The Bruce and Ruth Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology


JoVE 51937

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

 JoVE Immunology and Infection

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

1Unité de Génétique, Papillomavirus et Cancer Humain (GPCH), Institut Pasteur, 2Cellule Pasteur, Université Sorbonne Paris Cité, 3Center for Cancer Systems Biology (CCSB), Harvard Medical School, Department of Cancer Biology, Dana Farber Cancer Institute


JoVE 50404

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

 JoVE Chemistry

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), University of Heidelberg


JoVE 50839

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

 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

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

1Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas


JoVE 50436

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

 JoVE Biology

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

1Department Biologie I, Botanik, Ludwig-Maximilians-Universität, München


JoVE 51327

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

 JoVE Biology

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

1Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute


JoVE 3881

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 Biology

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

1GE Healthcare Bio-Sciences AB


JoVE 1746

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

 JoVE Immunology and Infection

Peptide-based Identification of Functional Motifs and their Binding Partners

1Department of Microbiology, Biochemistry, & Immunology, Morehouse School of Medicine, 2Institute for Systems Biology, 3Advanced Medical & Dental Institute, Universiti Sains Malaysia


JoVE 50362

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef’s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

 JoVE Biology

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

1Department of Biochemistry, University of Toronto, 2Department of Molecular Genetics, University of Toronto, 3Terrence Donnelly Centre for Cellular and Biomolecular Research (CCBR), University of Toronto


JoVE 1698

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

 JoVE Biology

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


JoVE 4057

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 Biology

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

1Max Planck Institute of Molecular Plant Physiology, 2University of Kaiserslautern


JoVE 4083

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

 JoVE Environment

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

1Lawrence Berkeley National Laboratory, The Molecular Foundry


JoVE 51087

More than half of proteins are small proteins (molecular mass <200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

 JoVE Biology

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

1Department of Genetics, Albert Einstein College of Medicine, 2Shanghai Key Laboratory of Complex Prescription and MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine


JoVE 51085

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

 JoVE Biology

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

1Promega Corporation, 2MS Bioworks LLC


JoVE 51553

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.  Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

 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 Biology

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

1Institute of Biochemistry, Stuttgart University


JoVE 52203

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

 JoVE Biology

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

1Department of Horticulture, University of Kentucky


JoVE 50685

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

 JoVE Immunology and Infection

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

1Genetics and Biochemistry Branch of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health


JoVE 51217

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution “snapshots” of an ordered assembly pathway in a living cell.

 JoVE Neuroscience

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

1Department of Neuroscience, Tufts University, 2Neuroscience Program, Tufts Sackler School of Graduate Biomedical Sciences


JoVE 4448

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

 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

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

1Department of Physics and Astronomy, Rowan University, 2Department of Biomedical and Translational Sciences, Rowan University, 3Department of Biomedical Sciences, Cooper Medical School of Rowan University, 4Department of Chemistry and Biochemistry, Rowan University


JoVE 50891

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

 JoVE Biology

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

1Institute of Molecular Medicine and Genetics, Georgia Health Sciences University


JoVE 2657

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

 JoVE Biology

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

1Department of Biochemistry / Analytics, caprotec bioanalytics GmbH, 2Institute of Organic Chemistry, RWTH Aachen University


JoVE 2264

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

 JoVE Biology

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

1Institute for Clinical Neurobiology, University of Wuerzburg, 2Department of Synapses - Circuits - Plasticity, Max Planck Institute of Neurobiology, Martinsried, 3Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians University of Munich


JoVE 50317

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

 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

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

1Department of Physics, University of Wisconsin - Milwaukee, 2Department of Biological Sciences, University of Wisconsin - Milwaukee


JoVE 2247

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of Förster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

 JoVE Biology

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

1Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, 2Department of Cell Biology, New York University School of Medicine


JoVE 3562

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

 JoVE Biology

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

1Architecture et Fonction des Macromolécules Biologiques (AFMB), Aix-Marseille Université, 2iBiTec-S, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), Commissariat à l'énergie atomique et aux énergies alternatives (CEA) Saclay, France


JoVE 51464

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

 JoVE Biology

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

1Department of Biochemistry & Molecular Biology, Wayne State University School of Medicine, 2Cardiovascular Research Institute, Wayne State University School of Medicine, 3Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine


JoVE 4091

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

 JoVE Biology

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

1Department of Biochemistry, University of Oxford


JoVE 50722

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

 JoVE Biology

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

1Department of Biological Sciences, Murray State University


JoVE 51387

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

 JoVE Biology

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

1Department of Physics and Astronomy, VU University Amsterdam


JoVE 51852

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

 JoVE Biology

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

1MRC LMCB, University College London, 2Center for Computational and Integrative Biology, Massachusetts General Hospital


JoVE 4382

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

 JoVE Bioengineering

High-throughput Protein Expression Generator Using a Microfluidic Platform

1The Mina & Everard Goodman Faculty of Life Sciences, The Nanotechnology Institute, Bar-Ilan University


JoVE 3849

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

 JoVE Biology

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


JoVE 3473

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 Biology

Reconstitution Of β-catenin Degradation In Xenopus Egg Extract

1Department of Cell and Developmental Biology and Program in Developmental Biology, Vanderbilt University Medical Center, 2Division of Gastroenterology, Hepatology & Nutrition and Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine


JoVE 51425

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

 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 Biology

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

1Medical Research Council, Clinical Sciences Centre, Imperial College, Hammersmith Hospital


JoVE 2631

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

 JoVE Biology

Analyzing Large Protein Complexes by Structural Mass Spectrometry

1Department of Biological Chemistry, Weizmann Institute of Science


JoVE 1954

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

 JoVE Bioengineering

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University


JoVE 3061

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

 JoVE Biology

In vitro Methylation Assay to Study Protein Arginine Methylation

1Department of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, 2Department of Hematology and Oncology, University of Illinois at Chicago, 3Jesse Brown Veterans Affairs Medical Center


JoVE 51997

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

 JoVE Bioengineering

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

1Center for Applied Proteomics and Molecular Medicine, George Mason University, 2Ceres Nanosciences


JoVE 51789

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

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