JoVE Application Notes
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.
Published July 15, 2011. Keywords: Protein transfer, turbo, rapid transfer, blotting, western blot, antigen, antibody, tank transfer, semi-dry, chemiluminescent, membrane
JoVE Application Notes
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.
Published August 6, 2012. Keywords: Advertisement, Protein transfer, turbo, rapid transfer, blotting, western blot, antigen, antibody, tank transfer, semi-dry, chemiluminescent, membrane
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
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.
Published November 20, 2014. Keywords: Basic Protocols, western blotting, fluorescent, LI-COR, protein, quantitative analysis, loading control
1Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven and Leuven Institute for Neuroscience and Disease (LIND)
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.
Published September 18, 2013. Keywords: Cellular Biology, biology (general), biochemistry, bioengineering (general), LRRK1, LRRK2, metabolic labeling, 32P orthophosphate, immunoprecipitation, autoradiography
1Integrated Science Center, Biology Department, The College of William & Mary
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.
Published October 30, 2013. Keywords: Basic Protocol, Life Sciences (General), budding yeast, protein extracts, bead beating, sumo, Ubiquitin, post-translational modifications, 6xHis affinity tag
1Diller Cancer Research Building, University of California, San Francisco
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.…
Published February 5, 2014. Keywords: Basic Protocol, Western blot, Bis-Tris, electrophoresis, dry blotting, protein transfer, infrared, Fluorescence, quantification, Antibody, Protein
JoVE Immunology and Infection
1Department Biologie, Friedrich-Alexander-Universität, 2Institut für Molekulare Pathogenese, Friedrich-Loeffler-Institut, 3Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen
The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.
Published June 25, 2015. Keywords: Infection, Apoptosis, Bax, Caspase 3, Coxiella burnetii, Doxycycline, Effector protein, Inducible expression, stable cell line, Tet system, Type IV Secretion System
1Physical Biosciences Division, Lawrence Berkeley National Laboratory
This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.
Published July 21, 2014. Keywords: Genetics, DNA-Affinity-Purified-chip, response regulator, transcription factor binding site, two component system, signal transduction, Desulfovibrio, lactate utilization regulator, ChIP-chip
1Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University
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.…
Published September 13, 2013. Keywords: Genetics, Receptors, G-Protein-Coupled, Protein Engineering, Signal Transduction, Biochemistry, Unnatural amino acid, site-directed mutagenesis, G protein-coupled receptor, targeted photocrosslinking, bioorthogonal labeling, targeted epitope tagging
1Department of Biological, Geological, and Environmental Sciences, Cleveland State University
Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.
Published June 10, 2015. Keywords: Biochemistry, Cell free in vitro transcription-translation, HeLa cell free expression, rhoptry proteins, mammalian cell free expression system, Plasmodium falciparum, Pro Bond affinity purification
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
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.
Published October 5, 2014. Keywords: Genetics, PRMT, protein methylation, SAMe, arginine, methylated proteins, methylation assay
1Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
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.…
Published July 5, 2014. Keywords: Biochemistry, two-electrode voltage-clamp, electrophysiology, biotinylation, Xenopus laevis oocytes, epithelial sodium channel, ENaC, proteases, proteolytic channel activation, ion channel, cleavage sites, cleavage fragments
1Department of Cell Biology, Scripps Institute
Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).
Published August 5, 2009. Keywords: Cellular Biology, FSM, qFSM, speckle, actin, cytoskeleton, fluorescence, microscopy, microinjection
1School of Materials Science and Engineering, Nanyang Technological University
Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats. …
Published June 11, 2015. Keywords: Bioengineering, Genetic engineering, protein expression, artificial extracellular matrix proteins, cell-binding domain, elastin-like domains, lower critical solution temperature, inverse transition cycling, phase transition behavior, protein purification
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
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).
Published August 21, 2011. Keywords: Neuroscience, Oligodendrocyte, myelination, in vitro, dorsal root ganglion neuron, co-culture, primary cells, mouse, neuroscience
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
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.
Published September 4, 2011. Keywords: Genetics, GPR54, KISS1R, precocious puberty, membrane receptor, proteasome, degradation, GC-rich, site-directed mutagenesis, immunoprecipitation
1Department of Biochemistry and Cell Biology, Stony Brook University, State University of New York
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.…
Published March 26, 2014. Keywords: Biochemistry, Ubiquitin/proteasome system, 26S proteasome, protein degradation, proteasome inhibitor, Western blotting, plant genetic transformation
1Department of Biochemistry and Cell Biology, State University of New York
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.
Published August 14, 2011. Keywords: Molecular Biology, protein-protein interactions, overlay, in vitro, western blotting, nitrocellulose membrane, insoluble protein
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
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.
Published May 18, 2009. Keywords: Neuroscience, Pull down, recombinant protein, GST, brain, rat, mass spectrometry, protein interactions, P2X2, macromolecular complex, channel, receptor, purinergic
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
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.
Published February 24, 2011. Keywords: Biochemistry, BN-PAGE, 2D gel electrophoresis, cellular lysate, dialysis, protein complex, multiprotein complex, protein interaction