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Isoelectric Focusing: Electrophoresis in which a pH gradient is established in a gel medium and proteins migrate until they reach the site (or focus) at which the pH is equal to their isoelectric point.

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

1Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, 2Hunan Engineering Laboratory for Structural Biology and Drug Design, Xiangya Hospital, Central South University, 3State Local Joint Engineering Laboratory for Anticancer Drugs, Xiangya Hospital, Central South University, 4The State Key Laboratory of Medical Genetics, Central South University

JoVE 56739


 Cancer Research

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

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

JoVE 51942


 Medicine

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

1Centro Cardiologico Monzino IRCCS, 2Cardiovascular Tissue Bank of Milan, Centro Cardiologico Monzino IRCCS, 3Department of Clinical Sciences and Community Health, Cardiovascular Section, University of Milan, 4Department of Cardiovascular Disease, Development and Innovation Cardiac Surgery Unit, Centro Cardiologico Monzino IRCCS

JoVE 55762


 Biochemistry

Separating Protein with SDS-PAGE

JoVE 5058

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

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

1Department of Chemical and Biomolecular Engineering, Ohio University, 2Biomedical Engineering Program, Russ College of Engineering and Technology, Ohio University, 3Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School

JoVE 51023


 Bioengineering

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

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

JoVE 51339


 Neuroscience

Two-Dimensional Gel Electrophoresis

JoVE 5686

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


 Biochemistry

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

1Center for Drug Evaluation and Research, Office of Product Quality, Office of Biotechnology Products, Division of Biotechnology Review and Research II, U.S. Food and Drug Administration

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JoVE 58947


 JoVE In-Press

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