Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Patent
1996-05-03
1999-03-30
Warden, Robert
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
G01N 2726, G01N 27447
Patent
active
058883657
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to methods for the separation of biological materials using polymeric materials.
BACKGROUND OF THE INVENTION
Purification and analysis of biological molecules is very often carried out by forcing these molecules to migrate through a gel. In gel electrophoresis the driving force is a voltage gradient across the gel and the gel matrix comprises natural or synthetic polymers. The synthetic polymers are usually formed by polymerization of double bonds present in monomer and cross-linker molecules.
Electrophoresis is based on the principle that charged molecules or substances will migrate when placed in an electric field. Since proteins and other biopolymers (e.g., DNA, RNA, enzymes and carbohydrates) are charged, they migrate at pH values other than their isoelectric point. The rate of migration depends, among other things, upon the charge density of the protein or biopolymer and the restrictive properties of the electrophoretic matrix. The higher the ratio of charge to mass, the faster the molecule will migrate. The more restrictive the medium, the more slowly an ion will migrate. Electrophoresis has the further advantage of generally requiring only very small (i.e., microgram or less) quantities of material for analysis.
Electrophoresis is generally performed in an aqueous solution or gel across which a voltage is applied. It is the voltage gradient that causes the migration of the species being separated. Gradients typically range from 10 volts/cm to many times higher, the magnitude depending on the nature of the separation being performed.
Many support media for electrophoresis are in current use. The most popular are sheets of paper or cellulose acetate, silica gels, agarose, starch and polyacrylamide. Paper, cellulose acetate, and thin layer silica materials are relatively inert and serve mainly for support and to minimize convection. Separation of proteins using these materials is based largely upon the charge density of the proteins at the pH selected.
On the other hand, starch, agarose and polyacrylamide gel materials not only minimize convection and diffusion but also actively participate in the separation process. These materials provide a porous medium in which the pore size can be controlled to approximate the size of the protein molecules being separated. In this way, molecular sieving occurs and provides separation on the basis of both charge density and molecular size.
The extent of molecular sieving is thought to depend on how closely the gel pore size approximates the size of the migrating particle. The pore size of agarose gels is sufficiently large that molecular sieving of most protein molecules is minimal and separation of proteins is based mainly on charge density. In contrast, polyacrylamide gels can have pores that more closely approximate the size of protein molecules and so contribute to the molecular sieving effect. Polyacrylamide has the further advantage of being a synthetic polymer which can be prepared in highly purified form.
The ability to produce gels having a wide range of polymer concentrations (and, therefore, since the gel network opening decreases with increasing polymer concentration, a wide range of controlled average pore size) as well as to form pore size gradients within the gels by virtue of polymer concentration gradients, are additional advantages of synthetic polymers such as polyacrylamide as electrophoresis gel media. Control over pore size enables mixtures of biological materials to be sieved on the basis of molecular size and enables molecular weight determinations to be performed. These determinations are especially accurate if proteins are treated with a detergent, such as sodium dodecyl sulfate (SDS), which neutralizes the effects of inherent molecular charge so that all SDS treated molecules, regardless of size, have approximately the same charge density values. This technique is referred to as SDS-PAGE (Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis).
Crosslinked polyacrylamide, produc
REFERENCES:
patent: 2976576 (1961-03-01), Wichterle et al.
patent: 3766047 (1973-10-01), Elevitch
patent: 3948743 (1976-04-01), Monthony et al.
patent: 4189370 (1980-02-01), Boschetti
patent: 4481094 (1984-11-01), Fernandez de Castro et al.
patent: 4657656 (1987-04-01), Ogawa
patent: 4695354 (1987-09-01), Sugihara et al.
patent: 4699705 (1987-10-01), Ogawa et al.
patent: 4704198 (1987-11-01), Ebersole et al.
patent: 4715942 (1987-12-01), Tezuka et al.
patent: 4737258 (1988-04-01), Ogawa et al.
patent: 4764164 (1988-08-01), Sasaki
patent: 4769408 (1988-09-01), Ogawa
patent: 4771089 (1988-09-01), Ofstead
patent: 4790919 (1988-12-01), Baylor, Jr.
patent: 4844786 (1989-07-01), Sugihara et al.
patent: 4857163 (1989-08-01), Gurske et al.
patent: 4859719 (1989-08-01), Ofstead
patent: 4891119 (1990-01-01), Ogawa
patent: 4948480 (1990-08-01), Christy, Jr. et al.
patent: 4963243 (1990-10-01), Ogawa et al.
patent: 4966792 (1990-10-01), Terai et al.
patent: 4985128 (1991-01-01), Ebersole et al.
patent: 5019232 (1991-05-01), Wilson et al.
patent: 5066376 (1991-11-01), Osterhoudt et al.
patent: 5114555 (1992-05-01), Stimpson
patent: 5143646 (1992-09-01), Nochumson et al.
patent: 5149416 (1992-09-01), Osterhoudt et al.
patent: 5164057 (1992-11-01), Mori et al.
patent: 5190629 (1993-03-01), Sugihara et al.
patent: 5196099 (1993-03-01), Mori et al.
patent: 5202007 (1993-04-01), Kozulic
patent: 5212253 (1993-05-01), Ponticello et al.
patent: 5238545 (1993-08-01), Yoshioka et al.
patent: 5290411 (1994-03-01), Zewert et al.
patent: 5338428 (1994-08-01), Zewert et al.
patent: 5397449 (1995-03-01), Zewert et al.
Zewert and Harrington, "Polyhydroxy and polyethyleneglycol (meth)acrylate polymers: Physical properties and general studies for their use as electrophoresis matrices" Electrophoresis 13:817-824 No month available (1992).
Zewert and Harrington, "Polyethyleneglycol methacrylate 200 as an electrophoresis matrix in hydroorganic solvents" Electrophoresis 13:824-831 No month available (1992).
Chen Sow-Hsin
Gandhi Khushroo
Liu Fu-Tong
Shih Lih-Bin
Applied Hydrogel Technology
Reiter Stephen E.
Starsiak Jr. John S.
Stewart Ramsey R.
Warden Robert
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