Electrophoresis, Polyacrylamide Gel:
Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.
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
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
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
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
1Department of Infectious Diseases, The Scripps Research Institute, 2Department of Chemistry, City College of New York
RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.
Published May 3, 2014. Keywords: Infectious Diseases, Cleavage, Polyadenylation, mRNA processing, Nuclear extracts, 3' Processing Complex
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 Biological Sciences, University of Alabama Huntsville, 2Department of Biology, Stanford University
A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.
Published February 25, 2011. Keywords: Molecular Biology, Ribosome stalling, ribosome isolation, peptidyl-tRNA, in vitro translation, RNA chemical modification, puromycin, antibiotics.
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