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  JoVE Biology

  
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  JoVE Neuroscience

  
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  JoVE Immunology and Infection

  
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  JoVE Clinical and Translational Medicine

  
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  JoVE Applied Physics

  
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  JoVE Behavior

  
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  JoVE Environment

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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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Model Organisms II

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Essentials of
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Essentials of Developmental Biology

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 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

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 Biology

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

1Division of Neurobiology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, 2Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, 3Centre for Integrative Physiology, University of Edinburgh, 4Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh


JoVE 52099

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

 JoVE Biology

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

1Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven and Leuven Institute for Neuroscience and Disease (LIND)


JoVE 50523

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

 JoVE Biology

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

1Integrated Science Center, Biology Department, The College of William & Mary


JoVE 50921

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

 JoVE Biology

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

1Diller Cancer Research Building, University of California, San Francisco


JoVE 51149

This protocol explores the latest advancements in performing Western blot analyses. These novel modifications employ a Bis-Tris gel system with a 35 min electrophoresis run time, a 7 min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

 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 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

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 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 Neuroscience

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

1Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Department of Pharmacological Sciences, Stony Brook University, 4Department of Medicine, University of Ottawa


JoVE 3324

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

 JoVE Biology

Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

1Division of Endocrinology and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, 2Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine


JoVE 2897

Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot.

 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 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

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

1Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, 3Department of Anesthesiology, Medicine and Physiology, David Geffen School of Medicine, University of California, Los Angeles


JoVE 1178

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

 JoVE Biology

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

1Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 2Centre for Biological Signalling Studies (bioss) and Biology III, Faculty of Biology, University of Freiburg, 3Department of Molecular Immunology, Max-Planck-Institute of Immunology and Epigenetics


JoVE 2164

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

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