Electrophoresis, Gel, Two-Dimensional:
Electrophoresis in which a second perpendicular electrophoretic transport is performed on the separate components resulting from the first electrophoresis. This technique is usually performed on polyacrylamide gels.
JoVE Immunology and Infection
1Department of Population Health, University of Georgia
We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.
Published July 22, 2011. Keywords: Immunology, PCR, Salmonella, multiplex, Serovar
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
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.
Published July 30, 2014. Keywords: Bioengineering, E. coli, expression, recombinant, high throughput (HTP), purification, auto-induction, immobilized metal affinity chromatography (IMAC), tobacco etch virus protease (TEV) cleavage, disulfide bond isomerase C (DsbC) fusion, disulfide bonds, animal venom proteins/peptides
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
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)
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 Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ)
Linear-amplification mediated (LAM)-PCR is a method developed to identify the exact positions of integrating viral vectors in the genome. The technique has evolved to be the superior method to study clonal dynamics in gene therapy patients, biosafety of novel vector technologies, T-cell diversity, cancer stem cell models, etc.
Published June 25, 2014. Keywords: Genetics, gene therapy, integrome, integration site analysis, LAM-PCR, retroviral vectors, lentiviral vectors, AAV, deep sequencing, clonal inventory, mutagenesis screen
1Research and Development, GE Healthcare Bio-Sciences AB
A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan™ DIGE technology.
Published November 26, 2008. Keywords: Biochemistry, Cell surface protein labelling, Ettan DIGE, CyDye DIGE Fluor minimal dyes, cell surface proteins, receptors, fluorescence, 2-D electrophoresis
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
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
1Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles
PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.
Published May 22, 2012. Keywords: Basic Protocols, PCR, optimization, primer design, melting temperature, Tm, troubleshooting, additives, enhancers, template DNA quantification, thermal cycler, molecular biology, genetics