Related Research Articles
Agarose is a heteropolysaccharide, generally extracted from certain red seaweed. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agarose is one of the two principal components of agar, and is purified from agar by removing agar's other component, agaropectin.
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.
Pharmacia was a pharmaceutical and biotechnological company in Sweden that merged with the American pharmaceutical company Upjohn in 1995.
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are commonly composed of dextran, agarose, or polyacrylamide polymers. The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.
Gel permeation chromatography (GPC) is a type of size-exclusion chromatography (SEC), that separates analytes on the basis of size, typically in organic solvents. The technique is often used for the analysis of polymers. As a technique, SEC was first developed in 1955 by Lathe and Ruthven. The term gel permeation chromatography can be traced back to J.C. Moore of the Dow Chemical Company who investigated the technique in 1964. The proprietary column technology was licensed to Waters Corporation, who subsequently commercialized this technology in 1964. GPC systems and consumables are now also available from a number of manufacturers. It is often necessary to separate polymers, both to analyze them as well as to purify the desired product.
A polyhistidine-tag is an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as hexa histidine-tag, 6xHis-tag, His6 tag, by the US trademarked name HIS TAG, and most commonly as His-Tag. The tag was invented by Roche, although the use of histidines and its vectors are distributed by Qiagen. Various purification kits for histidine-tagged proteins are available from Qiagen, Sigma, Thermo Scientific, GE Healthcare, Macherey-Nagel, Cube Biotech, Clontech, Bio-Rad, and others.
Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants won't interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.
Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.
Ion exchange is a reversible interchange of one kind of ion present in an insoluble solid with another of like charge present in a solution surrounding the solid with the reaction being used especially for softening or making water demineralised, the purification of chemicals and separation of substances.
Ion chromatography separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids. However, ion chromatography must be done in conditions that are one unit away from the isoelectric point of a protein.
Fast protein liquid chromatography (FPLC), is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid (the mobile phase) and a porous solid (the stationary phase). In FPLC the mobile phase is an aqueous solution, or "buffer". The buffer flow rate is controlled by a positive-displacement pump and is normally kept constant, while the composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs. The stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column. FPLC resins are available in a wide range of bead sizes and surface ligands depending on the application.
Sepharose is a tradename for a crosslinked, beaded-form of agarose, a polysaccharide polymer material extracted from seaweed. Its brand name is a portmanteau derived from Separation-Pharmacia-Agarose. A common application for the material is in chromatographic separations of biomolecules.
Chromatography is a physical method of separation that distributes the components you want to separate between two phases, one stationary, the other moving in a definite direction. Cold ethanol precipitation, developed by Cohn in 1946, manipulates pH, ionic strength, ethanol concentration and temperature to precipitate different protein fractions from plasma. Chromatographic techniques utilise ion exchange, gel filtration and affinity resins to separate proteins. Since the 1980s it has emerged as an effective method of purifying blood components for therapeutic use.
Electrochromatography is a chemical separation technique in analytical chemistry, biochemistry and molecular biology used to resolve and separate mostly large biomolecules such as proteins. It is a combination of size exclusion chromatography and gel electrophoresis. These separation mechanisms operate essentially in superposition along the length of a gel filtration column to which an axial electric field gradient has been added. The molecules are separated by size due to the gel filtration mechanism and by electrophoretic mobility due to the gel electrophoresis mechanism. Additionally there are secondary chromatographic solute retention mechanisms.
Diethylaminoethyl cellulose (DEAE-C) is a positively charged resin used in ion-exchange chromatography, a type of column chromatography, for the separation and purification of proteins and nucleic acids. Gel matrix beads are derivatized with diethylaminoethanol (DEAE) and lock negatively charged proteins or nucleic acids into the matrix. The proteins are released from the resin by increasing the salt concentration of the solvent or changing the pH of the solution as to change the charge on the protein.
Superose is a trade name for a collection of FPLC columns which are used in the automated separation of biological molecules. The different columns provided can separate a variety of macromolecules, ranging from small peptides and polysaccharides to DNA strands and entire viruses. The material inside the column is agarose based, meaning that it consists of sugars that are crosslinked to form a gel-like mass. The pores in this material have different sizes, and if a molecule is too big, it does not fit into the pores, meaning that it follows a shorter way to the end of the column.
Jerker Porath, was a Swedish biochemist who invented several separation methods for biomolecules. He was born in Sala.
BIA Separations is a biotechnology company focused on the production of methacrylate monolithic HPLC columns and developing industrial purification processes and analytical methods.
Desalting and buffer exchange are methods to separate soluble macromolecules from smaller molecules (desalting) or replace the buffer system used for another one suitable for a downstream application. These methods are based on gel filtration chromatography, also called molecular sieve chromatography, which is a form of size-exclusion chromatography. Desalting and buffer exchange are two of the most common gel filtration chromatography applications, and they can be performed using the same resin.
Anion-exchange chromatography is a process that separates substances based on their charges using an ion-exchange resin containing positively charged groups, such as diethyl-aminoethyl groups (DEAE). In solution, the resin is coated with positively charged counter-ions (cations). Anion exchange resins will bind to negatively charged molecules, displacing the counter-ion. Anion exchange chromatography is commonly used to purify proteins, amino acids, sugars/carbohydrates and other acidic substances with a negative charge at higher pH levels. The tightness of the binding between the substance and the resin is based on the strength of the negative charge of the substance.
References
- ↑ "From Sephadex to GE Healthcare". chemeurope.com. Archived from the original on July 31, 2012. Retrieved Jan 2, 2019.
- ↑ Porath, J; Flodin, P (1959). "Gel Filtration: A Method for Desalting and Group Separation". Nature . 183 (4676): 1657–1659. Bibcode:1959Natur.183.1657P. doi:10.1038/1831657a0. ISSN 1476-4687. PMID 13666849. S2CID 32287460.
- ↑ "Sephadex® ion exchange media" (PDF). chembio.uoguelph.ca. Pharmacia Biotech.
- ↑ "GE Healthcare Life Sciences - Instructions for Sephadex Media". .gelifesciences.com. Archived from the original on 2012-02-18. Retrieved 2011-12-02.
- ↑ GE Healthcare. "Sephadex LH-20" . Retrieved 2019-12-06.
- ↑ Hanukoglu I, Jefcoate CR (1980). "Pregnenolone separation from cholesterol using Sephadex LH-20 mini-columns". Journal of Chromatography A. 190 (1): 256–262. doi:10.1016/S0021-9673(00)85545-4. ISSN 0021-9673.
- ↑ "Instructions for Sephadex Media" (PDF). gelifesciences.co.jp (product manual). GE Healthcare. Apr 7, 2008. Retrieved Jan 2, 2019.