Electrophoresis, Polyacrylamide Gel:
Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.
1School of Biological Sciences, Nanyang Technological University, Singapore - NTU, 2Singapore-MIT Alliance for Reserach and Technology (SMART)
Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.
Published October 29, 2009. Keywords: Molecular Biology, DNA & RNA analysis, denaturing urea polyacrylamide gel electrophoresis, Protocols
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
JoVE Immunology and Infection
1Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 2The RNA Institute, SUNY Albany
This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.
Published January 19, 2015. Keywords: Immunology, HIV-1, Nucleocapsid protein, NCp7, TAR-RNA, DNA, oligonucleotides, annealing, Gel electrophoresis, NAME
1Biomedical Engineering Department, Saint Louis University
Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.
Published March 25, 2015. Keywords: Bioengineering, Multiwell, substrate stiffness, drug screening, polyacrylamide, Young’s modulus, high-throughput
1Team Alzheimer & Tauopathies, Jean-Pierre Aubert Research Centre, Inserm UMR 837, 2EA 4308-Department of Reproductive Biology-Spermiology-CECOS, CHRU-Lille, 3EA2686-Laboratorie d'Immunologie, Faculté de Médecine - Pôle Recherche, 4Department of Neurology, CHRU-Lille
A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.
Published April 10, 2014. Keywords: Neuroscience, proteomics, neurodegeneration, 2DE, human and mice brain tissue, fluorescence, immunoblotting.
Abbreviations: 2DE (two-dimensional gel electrophoresis), 2D-DIGE (two-dimensional fluorescence difference gel electrophoresis), mini-2DE (mini 2DE immunoblotting), IPG (Immobilized pH Gradients), IEF (isoelectrofocusing), AD (Alzheimer´s disease)
JoVE Immunology and Infection
1Department of Pediatrics, Centre for Understanding and Preventing Infection in Children, University of British Columbia
Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.
Published October 15, 2013. Keywords: Immunology, Bacteria, Aerobic, Gram-Negative Bacteria, Immune System Diseases, Respiratory Tract Diseases, Burkholderia, proteins, glass beads, 2-D gel electrophoresis
1Department of Microbial Genetics, Interfaculty Institute of Microbiology and Infection Medicine Tübingen (IMIT), Faculty of Science, University of Tübingen
We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.
Published October 31, 2014. Keywords: Molecular Biology, Primer extension, RNA mapping, 5' end, fluorescent primer, transcriptional starting point, TSP, RNase, toxin-antitoxin, cleavage site, gel electrophoresis, DNA isolation, RNA processing
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
1Department of Nanomedicine, Houston Methodist Research Institute, 2MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, 3Pediatrics Department of Union Hospital, Huazhong University of Science and Technology, 4CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience & Technology of China, 5Department of Medicine, Weill Cornell Medical College, 6Department of Cell and Developmental Biology, Weill Cornell Medical College
Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.
Published January 15, 2015. Keywords: Bioengineering, Porous silicon, siRNA, Nanodelivery system, Cancer therapy, Transfection, Polycation functionalization
1Lady Davis Institute for Medical Research, Jewish General Hospital, 2Department of Medicine, McGill University
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.
Published December 3, 2014. Keywords: Biochemistry, RNA metabolism, mRNA translation, post-transcriptional gene regulation, mRNA stability, IRE, IRP1, IRP2, iron metabolism, ferritin, transferrin receptor
1Laboratory of Structural Biology, NIEHS, National Institutes of Health
Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.
Published August 19, 2013. Keywords: Chemistry, Biochemistry, Genetics, Molecular Biology, Microbiology, Structural Biology, Chemical Biology, Eukaryota, Amino Acids, Peptides, and Proteins, Nucleic Acids, Nucleotides, and Nucleosides, Enzymes and Coenzymes, Life Sciences (General), enzymology, rapid quench-flow, active site titration, steady-state, pre-steady-state, single-turnover, kinetics, base excision repair, DNA glycosylase, 8-oxo-7, 8-dihydroguanine, 8-oxoG, sequencing
1RT Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research
High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.
Published May 31, 2013. Keywords: Genetics, Molecular Biology, Biochemistry, Virology, Cancer Biology, Medicine, Genomics, Nucleic Acid Probes, RNA Probes, RNA, High-throughput SHAPE, Capillary electrophoresis, RNA structure, RNA probing, RNA folding, secondary structure, DNA, nucleic acids, electropherogram, synthesis, transcription, high throughput, sequencing
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.
Published May 13, 2010. Keywords: Neuroscience, Cellular Biology, Aptamer, RNA, amyloid β-protein, oligomer, amyloid fibrils, protein assembly
JoVE Immunology and Infection
1Institute for Virology, Philipps-University Marburg
Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.
Published July 29, 2014. Keywords: Infectious Diseases, innate immune response, virus infection, pathogen recognition receptor, RIG-I, PKR, IRF-3, limited protease digestion, conformational switch, native PAGE, oligomerization
1Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles
A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.
Published April 20, 2012. Keywords: Genetics, Gel electrophoresis, agarose, DNA separation, ethidium bromide
1Institute for Molecular Biotechnology, Bio7, RWTH Aachen University, 2Fraunhofer Institute for Molecular Biology and Applied Ecology
Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.
Published June 13, 2014. Keywords: Environmental Sciences, heterologous expression, endoplasmic reticulum, endoglucanase, cellulose, glycosyl-hydrolase, fluorescence, cellulase, Trichoderma reesei, tobacco plants
1Department of Biology, University of Florida, 2UF Genetics Institute, University of Florida, 3Plant Molecular & Cellular Biology Program, University of Florida
Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4°C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.
Published February 12, 2012. Keywords: Basic Protocols, Molecular Biology, minigel, cassettes, protein, gel, electrophoresis
1School of Physics, Georgia Institute of Technology
This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.
Published June 28, 2014. Keywords: Molecular Biology, DNA looping, J factor, Single molecule, FRET, Gel mobility shift, DNA curvature, Worm-like chain
1Biological Medical Research Center (BMFZ), University Duesseldorf
This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.
Published August 3, 2009. Keywords: Basic Protocols, protein in-gel detection, Coomassie G-250, colloidal staining, 1- and 2-dimensional SDS-gels
JoVE Immunology and Infection
1Inserm UMR744, University Lille Nord de France
Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.
Published November 4, 2014. Keywords: Immunology, Biology, Human, Buffy coat, Monocytes, Macrophages, Culture, Proteins, Proteome, 2D DIGE-electrophoresis, 2D software
1Department of Chemistry and Biochemistry, University of Bern
The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.
Published April 3, 2014. Keywords: Chemistry, Nucleic acid analogues, Bioorganic Chemistry, PCR, primer extension reactions, organic synthesis, PAGE, HPLC, nucleoside triphosphates
1Medical Research Council (MRC), Cell Biology Unit and Laboratory for Molecular Cell Biology, University College London
Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.
Published September 3, 2011. Keywords: Molecular Biology, Polyacrilamyde Gel Electrophoresis (PAGE), inositol hexakisphosphate (IP6), phytic acid, diphosphoinositol pentakisphosphate (IP7), bisdiphoshoinositol tetrakisphosphate (IP8), IP6-kinase (IP6K), PP-IP5K, VIP1
1The Shraga Segal Department of Immunology, Microbiology & Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev
Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.
Published September 2, 2014. Keywords: Neuroscience, telomerase, telomeres, TRAP assay, PCR, gel electrophoresis, frontal lobe, cerebellum, brain stem
1Department of Chemistry, Hunter College, 2Department of Biochemistry, Albert Einstein College of Medicine
This protocol describes how to quantify the Mg(II)-dependent formation of RNA tertiary structure by two methods of hydroxyl radical footprinting.
Published October 17, 2011. Keywords: Molecular Biology, hydroxyl radical, footprinting, RNA, Fenton, equilibrium
1Center for Proteomics, Smith College, 2Department of Molecular Biophysics and Biochemistry, Yale University, 3Department of Chemistry, Smith College, 4Department of Biological Sciences and Center for Proteomics, Smith College
A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.
Published December 14, 2011. Keywords: Medicine, skeletal muscle, gender, 2-D gel electrophoresis, HPLC/MS, mouse
1Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University
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.
Published January 4, 2012. Keywords: Bioengineering, Gelatin Nanoparticle, Gene Therapy, Targeted Delivery, Pancreatic Cancer, Epidermal Growth Factor Receptor, EGFR
1Department of Biomedical Engineering, Duke University, 2Research Triangle MRSEC, Duke University
Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.
Published June 9, 2014. Keywords: Molecular Biology, elastin-like polypeptides, lower critical solution temperature, phase separation, inverse transition cycling, protein purification, batch purification
1Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University
A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.
Published July 6, 2012. Keywords: Genetics, Molecular Biology, Ribosome, Nascent polypeptide, Co-translational protein folding, Synonymous codon usage, gene regulation
1Oxford Protein Production Facility, Research Complex at Harwell, 2Protein Crystallography Group, Ruhr University, 3MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, 4Membrane Protein Laboratory, Diamond Light Source
A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.
Published January 6, 2015. Keywords: Microbiology, membrane proteins, green fluorescent protein, fluorescence detection, Escherichia coli, expression screening
1Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, 2Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, Texas A&M University, College Station
We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.
Published January 27, 2014. Keywords: Bioengineering, split-intein, self-assembly, shear-thinning, enzyme, immobilization, organic synthesis
1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 3School of Medicine, University of Washington
An in vitro method for preparing functional glucocorticoid receptor (GR)•hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.
Published September 21, 2011. Keywords: Biochemistry, glucocorticoid receptor, hsp90, molecular chaperone protein, in vitro reconstitution, steroid binding, biochemistry, immunoadsorption, immunoprecipitation, Experion, western blot
1Laboratory of Molecular Biology, Medical Research Council - MRC, 2European Bioinformatics Institute, EMBL Heidelberg, 3Computer and Information Science, University of Ljubljana, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute
The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.
Published April 30, 2011. Keywords: Cellular Biology, RNA biochemistry, transcriptome, systems biology, RNA-binding protein
1Department of Chemical and Biomolecular Engineering, University of Akron
Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.
Published January 22, 2014. Keywords: Bioengineering, protein engineering, recombinant protein production, AviTag, BirA, biotinylation, pET vector system, E. coli, inclusion bodies, Ni-NTA, size exclusion chromatography
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
1Stowers Institute for Medical Research, 2Department of Biochemistry & Molecular Biology, Kansas University Medical Center
This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.
Published October 23, 2014. Keywords: Biochemistry, chromatin remodeling, INO80, SNF2 family ATPase, structure-function, enzyme purification
1Department 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.
Published December 3, 2010. Keywords: Cellular Biology, multidrug resistance, membrane protein, purification, transmembrane transport, MexB, detergent solubilization, protein detergent complex
1Department of Biology, Ball State University, 2Division of Nephrology, Cincinnati Children's Hospital
This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.
Published February 16, 2015. Keywords: Molecular Biology, Ubiquitin-proteasome system, Saccharomyces cerevisiae, budding yeast, growth assay, protein extracts, western blotting, yeast genetics, mutants, endoplasmic reticulum-associated degradation, protein degradation
JoVE Immunology and Infection
1Department of Microbiology, Immunology, & Cancer Biology, University of Virginia Health System
We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.
Published May 28, 2012. Keywords: Immunology, Microbiology, Gram-negative, LPS, extraction, polysaccharide staining, Western immunoblot
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
JoVE Immunology and Infection
1Department of Biological Sciences, University of Alabama
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.
Published December 4, 2013. Keywords: Immunology, biology (general), environmental effects (biological, animal and plant), immunology, animal models, Immune System Diseases, Pathological Conditions, Signs and Symptoms, Life Sciences (General), Neuroinflammation, inflammation, nitric oxide, nitric oxide synthase, Drosophila, neurodegeneration, brain, Griess assay, nitrite detection, innate immunity, Parkinson disease, tissue culture
1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, 2Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, 3Department of Neurology, University of California, Los Angeles
Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid β-protein, and calcitonin, and a control peptide growth-hormone releasing factor.
Published January 12, 2009. Keywords: Cross-linking, PICUP, amyloid β-protein, oligomer, amyloid, protein assembly
1Biochemistry and Molecular Biology, Michigan State Universtiy
Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.
Published October 23, 2013. Keywords: Plant Biology, plant, long-distance transport, long-distance signaling, phloem, phloem exudate collection, assimilate transport, protein, RNA, lipids
1Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University
We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.
Published June 19, 2012. Keywords: Molecular Biology, Ribosome, nascent polypeptides, co-translational protein folding, translational arrest, in vitro translation
1University of California, San Diego
Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.
Published December 20, 2010. Keywords: Bioengineering, Decellularized, pericardium, extracellular matrix, in situ gelation, injectable, myocardial tissue engineering
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
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.
Published April 23, 2010. Keywords: JoVE Neuroscience, β-amyloid, oligomers, multimers, Western blotting, protein aggregation, Alzheimer's, antigen retrieval
1Department of Nanomedicine, The Methodist Hospital Research Institute, 2CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology
We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.
Published April 17, 2012. Keywords: Bioengineering, Nanoporous silica chip, Low molecular weight proteomics, Peptidomics, MALDI-TOF mass spectrometry, early diagnostics, proteomics
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 Zoology, University of British Columbia - UBC
The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.
Published February 9, 2009. Keywords: Immunology, influenza A virus, viral ribonucleoprotein, vRNP, glycerol gradient, negative staining, transmission electron microscopy
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
1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University
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.
Published July 18, 2011. Keywords: Bioengineering, atomic force microscopy, glucocorticoid receptor, protein-protein interaction, DNA-protein interaction, scanning probe microscopy, immunoadsorption