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
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 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
1Institute of Organic Chemistry, Department of Chemistry, RWTH Aachen University
DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.
Published November 22, 2014. Keywords: Biochemistry, S-adenosyl-l-methionine, AdoMet, SAM, aziridine cofactor, double activated cofactor, methyltransferase, DNA methylation, protein methylation, biotin labeling, fluorescence labeling, SMILing, mTAG
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