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You are here Biopharmaceutical glossary homepage > Technologies > Chromatography & electrophoresis Chromatography & electrophoresis
glossary & taxonomy Technologies term index Related glossaries include Bioprocessing Mass Spectrometry Proteins Proteomics Sequencing 2D See Two D affinity chromatography: A selective separation technique by which a compound (e.g., an antibody) is immobilized on a polymeric matrix and used to bind selectively other compounds. Following removal of the unattached components, the bound compound is displaced by changing the concentration of protons, salts, or cofactors in the eluent. IUPAC Biotech CE-MS (Capillary Electrophoresis- Mass Spectrometry): Separation is achieved through channels etched on the surface of the capillary (connected to an external high- voltage power supply) which delivers sample to ESI- MS. Automatable approach, with great sensitivity. CIEF Capillary electrophoresis IsoElectric Focusing: Sample is “focused” in the capillary tube, both separating and concentrating the protein or peptide at its isoelectric point. Then the entire mixture is delivered to the mass spectrometer. Using this technique the researchers have been able to record the molecular weight of all the proteins under study. Can be used to separate proteins from microorganisms. capillary array: A bundle of extremely narrow gel- filled tubes used to significantly speed the process of separating biological material. See capillary electrophoresis [ALSSA] capillary electromigration: Capillary electromigration techniques are becoming increasingly popular and important in chemical analysis, especially in the bioanalytical field. ... The separations in capillary electromigration techniques are achieved in narrow capillaries by employing a high electric field strength. These techniques include capillary electrophoretic techniques and electrically driven capillary chromatographic techniques, based on different separation principles. In some cases, these principles overlap. Capillary electromigration techniques have proved to be highly effective for the separation of small organic and inorganic ions, pharmaceuticals, explosives, dyes, polymers, proteins and peptides, DNA and RNA, cells, particles, etc. [5–10] IUPAC Recommendations, Terminology for Analytical Capillary Electromigration Techniques, 2003 http://www.iupac.org/publications/pac/2004/pdf/7602x0443.pdf Capillary Electrophoresis CE: A very sensitive separation technology, uses a capillary tube. Narrower terms: capillary gel electrophoresis, microcapillary electrophoresis capillary gel electrophoresis: Gel is in a capillary microchannel. Improved quantification for highly complex samples. Related term: microcapillary electrophoresis. carrier ampholyte(s): Small soluble molecules that are capable of acting as an acid or a base in standard ISO-DALT 2D gel electrophoresis. chromatography: A physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. IUPAC Compendium Techniques used to separate mixtures of substances based on differences in the relative affinities of the substances for mobile and stationary phases. A mobile phase (fluid or gas) passes through a column containing a stationary phase of porous solid or liquid coated on a solid support. Usage is both analytical for small amounts and preparative for bulk amounts. MeSH, 1966 DGGE (Denaturing Gradient Gel Electrophoresis): A technique for screening for SNPs. dHPLC denaturing high- performance liquid chromatography: Used in SNP- detection methods based on discrimination between perfect and mismatched hybridization, measures melting temperatures. (Mismatching results in lowered melting temperature.) This method involves passing the hybridization mixture through a chromatographic column several times at different temperatures and deriving the melting temperature from changes in the chromatogram. This method has the advantage of not needing labeled components; however, each assay requires at least 15 minutes to complete. dielectrophoresis: http://en.wikipedia.org/wiki/Dielectrophoresis Edman degradation: A lab technique used to find out the order of amino acids in a polypeptide (chain of amino acids). It involves using the Edman reagent, phenyl isothiocyanate (PITC), to react one by one with each amino acid, in order. The technique is used in machines which automatically sequence (determine the order of subunits) polypeptides. [OMD] electrophoresis: The motion of colloidal particles in an electric field. IUPAC Compendium Technique for separating and purifying molecules according to the relative distance they travel under the influence of an electric current. [NHLBI] An electrochemical process in which macromolecules or colloidal particles with a net electric charge migrate in a solution under the influence of an electric current. MeSH A method of separating large molecules (such as DNA fragments or proteins) from a mixture of similar molecules. An electric current is passed through a medium containing the mixture, and each kind of molecule travels through the medium at a different rate, depending on its electrical charge and size. Separation is based on these differences. Agarose and acrylamide gels are the media commonly used for electrophoresis of proteins and nucleic acids. [DOE] A technique for separating and analyzing substances based on the movement of their ions in an electrical field. Related term: gel electrophoresis Expanded Bed Adsorption EBA: What chromatographers need is a method that combines sample preparation with the first stage of chromatography. Welcome to expanded- bed adsorption (EBA) chromatography. EBA uses apparatus that is familiar to most users of standard liquid chromatography. The column has a flow adapter that is positioned to suit the specific step of resin preparation or protein purification. And a series of pumps and valves, connected through the adapter and bottom of the column, control the flow rate and direction of the buffer and sample loading (1, 2). Thus, it is feasible to perform preliminary EBA trials with a little ingenuity and standard chromatographic equipment. ...In the more traditional packed-bed methods, where the resin is confined between the bottom of the column and the flow adapter, clogging occurs when particulate matter and cell debris cannot flow around the closely packed resin beads. In contrast, EBA columns are fed from below, and the adapter is held away from the packed resin level, giving the resin room to expand and thus creating spaces between the beads. Randall C. Willis "Expanded Bed Adsorption" Modern Drug Discovery 4 (12): 43- 44 Dec. 2001 http://pubs.acs.org/subscribe/journals/mdd/v04/i12/html/12toolbox.html frontal affinity chromatography: Method for screening mixtures of compounds for affinity against an immobilised target. IUPAC COMBINATORIAL CHEMISTRY gel electrophoresis: A DNA separation technique that is very important in DNA sequencing. Standard sequencing procedures involve cloning DNA fragments into special sequencing cloning vectors that carry tiny pieces of DNA. The next step is to determine the base sequence of the tiny fragments by a special procedure that generates a series of even tinier DNA fragments that differ in size by only one base. These nested fragments are separated by gel electrophoresis, in which the DNA pieces are added to a gelatinous solution, allowing the fragments to work their way down through the gel. Smaller pieces move faster and will reach the bottom first. Movement through the gel is hastened by applying an electrical field to the gel. [DOE] Electrophoresis performed in a polymer matrix (gel) Broader term: electrophoresis, PAGE, SDS- PAGE Narrower terms: 2D gel electrophoresis, pulsed- field gel electrophoresis gel shift: A method by which the interaction of a nucleic acid (DNA or RNA) with a protein is detected. The mobility of the nucleic acid is monitored in an agarose gel in the presence and absence of the protein: if the protein binds to the nucleic acid, the complex migrates more slowly in the gel (hence "gel shift"). A "supershift" allows determination of the specific protein, by virtue of a second shift in mobility that accompanies binding of a specific antibody to the nucleic acid-protein complex. Lemon, Stanley M. and Alan Barbour "Glossary of terms commonly used in molecular biology" UNC- Chapel Hill and Univ. of Texas Health Science Center, US, 2000 http://www.med.unc.edu/wrkunits/3ctrpgm/pmbb/mbt/GLOS.htm glass capillary electrophoresis: . A type of automated sequencer based on glass capillary electrophoresis. Each automated sequencer possesses an array of capillaries that contain the DNA separation matrix. Fluorescently labeled DNA is applied by electrokinetic injection into each capillary, resolved by electrophoresis through the capillary matrix, and then detected as it passes by a clear window in each capillary or as the DNA fragments exit the end of each capillary. Before the next run, the instrument completely recycles itself, injecting new separation matrix into each capillary and preparing the instrument for data collection. HPLC High Performance Liquid Chromatography:
Liquid chromatographic
techniques which feature high inlet pressures, high sensitivity, and high
speed. MeSH, 1981 IEF (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. MeSH, 1974 First reported in 1961. Earliest version used carrier ampholytes; newer uses immobilized pH gradients. Compare with yeast two hybrid. Proteomics IPG (Immobilized pH Gradient). Now standard 2D gel electrophoresis method. Amersham Pharmacia led the commercialization effort. Can be used for pH range 3-12 and is method of choice for IsoElectric Focusing. Narrower term: immobilized pH gradient shift. IPG-DALT: Immobilized pH Gradient with protein mass expressed in Daltons. ISO-DALT: ISOelectric focusing electrophoresis, measured in Daltons. Cannot resolve basic proteins with pI > 7.0-7.5 pH. Use of isoelectric focusing greatly improves resolution. immobilized pH gradient shift: Introduction of gels with immobilized pH gradient (in 1982) improved the separability of 2D gel electrophoresis. Narrower term: IPG. immobilized pH gradient strips, very narrow range: Also called “zoom gels” or proteomic contigs, used to improve sensitivity of 2D gel electrophoresis. Broader term IPG. immunoprecipitation: Antibody based affinity purification. in gel protein digestion: A method to cleave proteins into more easily identifiable peptide subunits directly in the gel immediately following separation of the proteins by gel electrophoresis. This method uses different enzymes, which specifically cleave between certain amino acid sequences. ALSSA isoelectric point: The pH in solutions of proteins and related compounds at which the dipolar ions are at a maximum. MeSH, 1991 The pH value at which the net electric charge of an elementary entity is zero. pI is a commonly used symbol for this kind- of- quantity. It should be replaced by pH (I) because it is a pH determined under that particular condition. IUPAC Compendium liquid chromatography LC: A separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or on a plane. Present- day liquid chromatography generally utilizing very small particles and a relatively high inlet pressure is often characterized by the term high- performance (or high- pressure) liquid chromatography, and the acronym HPLC. IUPAC Compendium Chromatographic techniques in which the mobile phase is a liquid. MeSH, 1976 microcapillary electrophoresis (micro-capillary electrophoresis): Is this different from microchip electrophoresis? microscale HPLC systems: Nanoscience & miniaturization glossary multidimensional chromatography: The use of two or more columns or chromatographic techniques which enable a better separation. It is useful for sample cleanup, increased resolution, and increased throughput. It can be used off- line by collecting fractions and reinjecting onto a second column or on- line by the use of a switching valve. It also called coupled column chromatography, column switching, multicolumn chromatography, or boxcar chromatography. HPLC Glossary, Waters Corp., 2003 http://www.waters.com/watersdivision/images/aboutus/hplcglossary.htm#M NEPHGE: Non-Equilibrium PH Gradient Electrophoresis. Nonequilibrium
pH gel electrophoresis (NEPHGE) is a technique developed to resolve proteins
with extremely basic isoelectric points (pH 7.5-11.0) (1,2).
These proteins are difficult to resolve using standard IEF, because the presence
of urea in IEF gels has a buffering effect and prevents the pH gradient from
reaching the very basic values (with a pH above 7.3-7.6) (3). In addition,
cathodic drift causes many very basic proteins to run off the end of the gel.
Mary Lopez The Protein Protocols Handbook
2002, Part II, 181-183,
DOI: 10.1385/1-59259-169-8:181
non-denaturing gel electrophoresis: DNA fragments separate in a double stranded form. Also known as “native gel” systems. Related term: IsoElectric Focusing. pI: See isoelectric point. PAGE PolyAcrylamide Gel Electrophoresis: Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. [Metathesaurus] Narrower term: SDS-PAGE. proteomic contigs: See immobilized pH gradient strip, very narrow range. pulsed field gel electrophoresis: Electrophoresis in which the direction of the electric field is changed periodically. This technique is similar to other electrophoretic methods normally used to separate double- stranded DNA molecules ranging in size up to tens of thousands of base-pairs. However, by alternating the electric field direction one is able to separate DNA molecules up to several million base- pairs in length. MeSH, 1992 SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis): May be helped by mass spectrometry, can be blotted or eluted for further chemical analysis i.e. Edman degradation, amino acid microsequencing or scanned MALDI-MS. Also known as denaturing gel electrophoresis or native gel electrophoresis. [Glick] Broader terms: gel electrophoresis, PAGE. Supercritical Fluid Chromatography SFC: A chromatography method using supercritical fluid, usually carbon dioxide under very high pressure (around 73 atmospheres or 1070 psi at room temperature) as the mobile phase. Other solvents are sometimes added as modifiers. This is used both for analytical (SFC) and extraction (SFE) purposes. MeSH, 2002 tandem chromatography: Tandem chromatography is used to automatically collect selected fractions from one column and inject them onto a second column. Eluted fractions from the first column are collected into a DynaLoop or other sample loop. After injection of the collected sample, it is eluted from the second column. ... This automated procedure can be performed at neutral pH, as an alternative to affinity chromatography, which requires low-pH buffers to elute the antibodies. Basic Principles of Chromatography, Dept of Chemistry, Minnesota State University Moorhead http://www.mnstate.edu/biotech/FPLC_talk_hndout.pdf Two D DIGE: 2D Differential (or Difference) In Gel Electrophoresis Two D gel electrophoresis: Electrophoresis in which a second perpendicular electrophoretic transport is performed on the separate components resulting from the first electrophoresis. This technique is usually performed on polyacrylamide gels. MeSH, 1989 Proteins are first separated across a gel according to their isoelectric point, then separated in a perpendicular direction on the basis of their molecular weight. First introduced in 1975. Most commonly used method for protein separation in proteomics. Two-Dimensional Difference Gel Electrophoresis: Methods of comparing two or more samples on the same two-dimensional gel electrophoresis gel. MeSH 2011 zoom gels: See immobilized pH gradient strips, very narrow range.
Chromatography & Electrophoresis resources IUPAC definitions are reprinted with the permission of the International Union of Pure and Applied Chemistry.
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