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Isoelectric Point: The pH in solutions of proteins and related compounds at which the dipolar ions are at a maximum.
 Science Education: Essentials of Organic Chemistry

Separation of Mixtures via Precipitation

JoVE Science Education

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

 JoVE Medicine

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

 JoVE Neuroscience

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

 Science Education: Essentials of Analytical Chemistry

Ion-Exchange Chromatography

JoVE Science Education

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

 JoVE Chemistry

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

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


JoVE 51183

 Science Education: Essentials of Biochemistry

Two-Dimensional Gel Electrophoresis

JoVE Science Education

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

 JoVE In-Press

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

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

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JoVE 54785

 JoVE Biology

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

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


JoVE 52099

 JoVE Behavior

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

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


JoVE 52899

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 Science Education: Basic Methods in Cellular and Molecular Biology

Separating Protein with SDS-PAGE

JoVE Science Education

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.

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 JoVE Bioengineering

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

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 Science Education: Essentials of Environmental Science

Tree Survey: Point-Centered Quarter Sampling Method

JoVE Science Education

A number of methods are available for sampling forest communities. Point-centered quarter is one such method. It is used to gather information on the density, frequency, and coverage of tree species found in a forest. This information provides the ability to estimate the number of individual trees encountered, how often a certain tree occurs, how common the tree is compared to other trees, and the size of the tree. Compared to the standard plot analysis, the point-centered quarter method is more efficient, which is a major advantage. In a fixed-area plot sampling, a small portion of the total area of the forest is examined. In this small subsample, the density is determined directly by counting and identifying each tree. The ratio between size of the subplot and the overall forest size is used to determine the density for the entire forest.

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 Science Education: Essentials of General Chemistry

Freezing-Point Depression to Determine an Unknown Compound

JoVE Science Education

Source: Laboratory of Lynne O' Connell — Boston College

When a solid compound is dissolved in a solvent, the freezing point of the resulting solution is lower than that of the pure solvent. This phenomenon is known as freezing-point depression, and the change in temperature is directly related to the molecular weight of the solute. This experiment is designed to find the identity of an unknown compound by using the phenomenon of freezing-point depression to determine its molecular weight. The compound will be dissolved in cyclohexane, and the freezing point of this solution, as well as that of pure cyclohexane, will be measured. The difference between these two temperatures allows for the calculation of the molecular weight of the unknown substance.

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