1Inserm U1127, 2CNRS UMR 7225, 3UPMC Univ Paris 06, UMR S 1127, Sorbonne Universités, 4Institut du Cerveau et de la Moelle épinière (ICM)
1Radiation Oncology, University of California, Davis
1Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, 2James H. Quillen Veterans Affairs Medical Center
Source: Laboratory of Dr. Ana J. García-Sáez — University of Tübingen
Most samples of interest are mixtures of many different components. Sample preparation, a key step in the analytical process, removes interferences that may affect the analysis. As such, developing separation techniques is an important endeavor not just in academia, but also in industry.
One way to separate mixtures is to use their solubility properties. In this short paper, we will deal with aqueous solutions. The solubility of a compound of interest depends on (1) ionic strength of solution, (2) pH, and (3) temperature. By manipulating with these three factors, a condition in which the compound is insoluble can be used to remove the compound of interest from the rest of the sample.1…
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
1BioMedomics Inc., 2Division of Hematology, Department of Medicine, Duke University Medical Center
1Division of Molecular Oncology, IRCCS, San Raffaele Scientific Institute, 2Department of Haemato-Oncology, King's College London, 3IFOM, FIRC Institute of Molecular Oncology, 4Università Vita-Salute San Raffaele
Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Ion-exchange chromatography is a type of chromatography that separates analytes based on charge. A column is used that is filled with a charged stationary phase on a solid support, called an ion-exchange resin. Strong cation-exchange chromatography preferentially separates out cations by using a negatively-charged resin while strong anion-exchange chromatography preferentially selects out anions by using a positively-charged resin. This type of chromatography is popular for sample preparation, for example in the cleanup of proteins or nucleic acid samples.
Ion-exchange chromatography is a two-step process. In the first step, the sample is loaded onto the column in a loading buffer. The binding of the charged sample to the column resin is based on ionic interactions of the resin to attract the sample of the opposite charge. Thus, charged samples of opposite polarity to the resin are strongly bound. Other molecules that are not charged or are of the opposite charge are not bound and are washed through the column. The second step is to elute the analyte that is bound to the resin. This is accomplished with a salt gradient, where the amount of salt in the buffer is slowly increased. Fractions are collected at the end of the column as…
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
1Department of Bioengineering, Stanford University, 2Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, 3Campus de Cantoblanco, Universidad Autonoma de Madrid, 4Department of Microbiology and Immunology, Stanford University School of Medicine
1Inserm UMR744, University Lille Nord de France
Immunology and Infection
Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially a…
1Unidad de Investigación y Desarrollo, Probiomed S.A. de C.V.
1Institute of Nanoengineering and Microsystems, National Tsing Hua University, 2Taichung Veterans General Hospital
1Savannah River Consulting, L.L.C., 2Savannah River National Laboratory
1Biomedical Engineering Department, University of Connecticut, 2Mechanical Engineering Department, University of Connecticut
1Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, 2Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México
1UMR8576, CNRS, Lille University, 2UMR-S1172, INSERM CNRS, Lille University
1Department of Biochemistry and Biomedical Sciences, McMaster University, 2Department of Chemistry and Chemical Biology, McMaster University, 3Department of Medicine, McMaster University
1Department of Chemical and Biomolecular Engineering, University of Akron
1Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University
1Division of Virology, Department of Pathology, University of Cambridge
1Bristol Centre for Functional Nanomaterials, University of Bristol, 2Department of Materials, Imperial College London, 3Self Assembly Group, CIC nanoGUNE, 4Ikebasque, Basque Foundation for Science, 5School of Cellular and Molecular Medicine, University of Bristol, 6H.H. Wills Physics Laboratory, University of Bristol
1Center for Applied Proteomics and Molecular Medicine, George Mason University, 2Ceres Nanosciences
1BioNano Laboratory, School of Engineering, University of Guelph
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
1National Exposure Research Laboratory, U.S. Environmental Protection Agency
1Department of Neurology, The Ohio State University Wexner Medical Center, 2Department of Physical Medicine and Rehabilitation, The Ohio State University, 3Department of Neuroscience, The Ohio State University Wexner Medical Center, 4Department of Biochemistry and Pharmacology, The Ohio State University Wexner Medical Center
1Structure et Propriétés d'Architectures Moléculaires, Institut Nanosciences et Cryogénie, CEA-Grenoble, 2Institut de Chimie Moléculaire et des Matériaux d'Orsay, Université Paris-Sud, 3Institut de Biologie Structurale
1Genome Stability Laboratory, Oncology Axis, Hôtel-Dieu de Québec
1Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture
1Department of Chemical Engineering, Michigan Technological University, 2Department of Mechanical Engineering, Michigan Technological University, 3XG Sciences, Inc.
Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their size and nothing else. SDS, an anionic detergent, is used to produce an even charge across the length of proteins that have been linearized. By first loading them into a gel made of polyacrylamide and then applying an electric field to the gel, SDS-coated proteins are then separated. The electric field acts as the driving force, drawing the SDS coated proteins towards the anode with larger proteins moving more slowly than small proteins. In order to identify proteins by size, protein standards of a known size are loaded along with samples and run under the same conditions.
This video presents an introduction to SDS-PAGE by first explaining the theory behind it and later demonstrating its step-by-step procedure. Various experimental parameters, such as the polyacrylamide concentration and voltage applied to the gel are discussed. Downstream staining methods like Coomassie and silver stains are introduced, and variations of the method, like 2D gel electrophoresis are presented.…
Basic Methods in Cellular and Molecular Biology