SDS PAGE
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(New page: '''INTRODUCTION''' Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the ...) |
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'''INTRODUCTION''' | '''INTRODUCTION''' | ||
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Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the nett charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique. | Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the nett charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique. | ||
'''Support Matrices''' | '''Support Matrices''' | ||
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Generally the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage. | Generally the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage. | ||
In addition, the most commonly used support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Because dilute agarose gels are generally more rigid and easy to handle than polyacrylamide of the same concentration, agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes. Polyacrylamide, which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation. | In addition, the most commonly used support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Because dilute agarose gels are generally more rigid and easy to handle than polyacrylamide of the same concentration, agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes. Polyacrylamide, which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation. | ||
| - | Separation of Proteins and Nucleic Acids | + | '''Separation of Proteins and Nucleic Acids''' |
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Proteins are amphoteric compounds; their nett charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a nett negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The nett charge carried by a protein is in addition independent of its size - ie: the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules. | Proteins are amphoteric compounds; their nett charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a nett negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The nett charge carried by a protein is in addition independent of its size - ie: the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules. | ||
Nucleic acids however, remain negative at any pH used for electrophoresis and in addition carry a fixed negative charge per unit length of molecule, provided by the PO4 group of each nucleotide of the the nucleic acid. Electrophoretic separation of nucleic acids therefore is strictly according to size. | Nucleic acids however, remain negative at any pH used for electrophoresis and in addition carry a fixed negative charge per unit length of molecule, provided by the PO4 group of each nucleotide of the the nucleic acid. Electrophoretic separation of nucleic acids therefore is strictly according to size. | ||
'''SDS- PAGE OF PROTEINS''' | '''SDS- PAGE OF PROTEINS''' | ||
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Separation of Proteins under Denaturing conditions | Separation of Proteins under Denaturing conditions | ||
Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by "wrapping around" the polypeptide backbone - and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length - ie: the denatured polypeptides become "rods" of negative charge cloud with equal charge or charge densities per unit length. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size: this is done with 2- mercaptoethanol or dithiothreitol. In denaturing SDS-PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight. | Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by "wrapping around" the polypeptide backbone - and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length - ie: the denatured polypeptides become "rods" of negative charge cloud with equal charge or charge densities per unit length. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size: this is done with 2- mercaptoethanol or dithiothreitol. In denaturing SDS-PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight. | ||
'''Determination of Molecular Weight''' | '''Determination of Molecular Weight''' | ||
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This is done by SDS-PAGE of proteins - or PAGE or agarose gel electrophoresis of nucleic acids - of known molecular weight along with the protein or nucleic acid to be characterised. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide, or native nucleic acid, and its Rf. The Rf is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye-front. A simple way of determining relative molecular weight by electrophoresis (Mr) is to plot a standard curve of distance migrated vs. log10MW for known samples, and read off the logMr of the sample after measuring distance migrated on the same gel. | This is done by SDS-PAGE of proteins - or PAGE or agarose gel electrophoresis of nucleic acids - of known molecular weight along with the protein or nucleic acid to be characterised. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide, or native nucleic acid, and its Rf. The Rf is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye-front. A simple way of determining relative molecular weight by electrophoresis (Mr) is to plot a standard curve of distance migrated vs. log10MW for known samples, and read off the logMr of the sample after measuring distance migrated on the same gel. | ||
'''Continuous and Discontinuous Buffer Systems''' | '''Continuous and Discontinuous Buffer Systems''' | ||
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There are two types of buffer systems in electrophoresis, continuous and discontinuous. A continuous system has only a single separating gel and uses the same buffer in the tanks and the gel. In a discontinuous system, a non-restrictive large pore gel, called a stacking gel, is layered on top of a separating gel called a resolving gel. Each gel is made with a different buffer, and the tank buffers are different from the gel buffers. The resolution obtained in a discontinuous system is much greater than that obtained with a continuous system. | There are two types of buffer systems in electrophoresis, continuous and discontinuous. A continuous system has only a single separating gel and uses the same buffer in the tanks and the gel. In a discontinuous system, a non-restrictive large pore gel, called a stacking gel, is layered on top of a separating gel called a resolving gel. Each gel is made with a different buffer, and the tank buffers are different from the gel buffers. The resolution obtained in a discontinuous system is much greater than that obtained with a continuous system. | ||
Revision as of 05:23, 9 September 2008
INTRODUCTION
Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the nett charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique.
Support Matrices
Generally the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage. In addition, the most commonly used support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Because dilute agarose gels are generally more rigid and easy to handle than polyacrylamide of the same concentration, agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes. Polyacrylamide, which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation.
Separation of Proteins and Nucleic Acids
Proteins are amphoteric compounds; their nett charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a nett negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The nett charge carried by a protein is in addition independent of its size - ie: the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules. Nucleic acids however, remain negative at any pH used for electrophoresis and in addition carry a fixed negative charge per unit length of molecule, provided by the PO4 group of each nucleotide of the the nucleic acid. Electrophoretic separation of nucleic acids therefore is strictly according to size.
SDS- PAGE OF PROTEINS
Separation of Proteins under Denaturing conditions Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by "wrapping around" the polypeptide backbone - and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length - ie: the denatured polypeptides become "rods" of negative charge cloud with equal charge or charge densities per unit length. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size: this is done with 2- mercaptoethanol or dithiothreitol. In denaturing SDS-PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight.
Determination of Molecular Weight
This is done by SDS-PAGE of proteins - or PAGE or agarose gel electrophoresis of nucleic acids - of known molecular weight along with the protein or nucleic acid to be characterised. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide, or native nucleic acid, and its Rf. The Rf is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye-front. A simple way of determining relative molecular weight by electrophoresis (Mr) is to plot a standard curve of distance migrated vs. log10MW for known samples, and read off the logMr of the sample after measuring distance migrated on the same gel.
Continuous and Discontinuous Buffer Systems
There are two types of buffer systems in electrophoresis, continuous and discontinuous. A continuous system has only a single separating gel and uses the same buffer in the tanks and the gel. In a discontinuous system, a non-restrictive large pore gel, called a stacking gel, is layered on top of a separating gel called a resolving gel. Each gel is made with a different buffer, and the tank buffers are different from the gel buffers. The resolution obtained in a discontinuous system is much greater than that obtained with a continuous system.
