Gel electrophoresis is a group of techniques used by scientists to separate molecules based on physical characteristics such as size, shape, or isoelectric point. Gel electrophoresis is usually performed for analytical purposes, but may be used as a preparative technique to partially purify molecules prior to use of other methods such as mass spectrometry, Polymerase chain reaction, cloning, DNA sequencing, or immuno-blotting for further characterization. The first part, "gel", refers to the matrix used to separate the molecules. In most cases the gel is a crosslinked polymer whose porosity can be controlled by the scientist. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually made with different concentrations of acrylamide and a cross-linker, producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a few hundred base (chemistry)), the preferred matrix is purified agarose. In both cases, the gel forms a solid but porous matrix that looks and feels like clear jello. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and needs to be handled using Good Laboratory Practices (GLP) to avoid poisoning. The second part, "electrophoresis", refers to the electromotive force (EMF) that is used to push or pull the molecules through the gel matrix; by placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, towards the anode if negatively charged or towards the cathode if positively charged (note that gel electrophoresis operates as an electrolytic cell; the anode is positive and the cathode is negative). In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the natural negative charge carried on their sugar-phosphate backbone. Double-stranded DNA fragments natually behave as long rods, so their migration through the gel is relative to their radius of gyration, or, roughly, size. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to renature the nucleic acids and cause them to behave as long rods again. Proteins, on the other hand, can have different charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are ususally denatured in the presense of a detergent such as sodium dodecyl sulfate/sodium docecyl phosphate (SDS/SDP) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the size of the protein, so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like they were long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape. After the electrophoresis run, when the smallest molecules have almost reached the anode, the molecules in the gel can be staining (biology) to make them visible. Ethidium bromide, Silver, or Coomassie blue dye can be used. Other methods can also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules luminescence under ultraviolet light, a photograph can be taken of the gel under ultraviolet light. If the molecules to be separated contain radioactivity atoms, an isotopic tracer can be recorded of the gel (as in the example shown above). If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components. Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are special markers available - ladders - which contain a mxiture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule. ==Types== Gel electrophoresis is used in molecular biology, genetics, and biochemistry: *Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis. See the "Chain termination method" page for an example of a Acrylamide DNA sequencing gel. *Gel electrophoresis of proteins is usually done in an sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), by native gel electrophoresis, or by 2-D electrophoresis. *Capillary electrophoresis *DNA electrophoresis *Zymography ==See also== * Protein electrophoresis * Electrofocusing * Southern blotting * Northern blotting * Western blotting ==External links== * [http://gslc.genetics.utah.edu/units/biotech/gel/ Biotechniques Laboratory electrophoresis demonstration], from the University of Utah's Genetic Science Learning Center * [http://www.westernblotting.org Protein electrophoresis and Western Blotting] Molecular biologyBiochemistryAnalytical chemistry Laboratory techniques
Umm, this is mostly about molecules, not about electrophoresis... -- user:Marj Tiefert == Gel electrophoresis =/ electrophoresis == USP<726> cites Gel, Disk, Zone among others. Also, USP<727> Capillary Electrophoresis, as well as Capillary Zone electrophoresis. Perhaps electrophoresis should not redirect here. Eh, I'm an idiot, although I do still think Gel electrophoresis is still too specific for electrophoresis to redirect here.User:Ix 01:47, 31 May 2004 (UTC) ---- Its not very accurate either. Normally electrophoresis of marcomolecules separates based on size and shape, rather than charge, or at least that's the way it is used. You can also separate based upon the isoelectric point. I think it would be worthwhile redoing the article. This probably should be one place that electrophoresis is linked, but surely not the only one. User:Srlasky 22:43, 2004 Oct 24 (UTC) == Electrophoresis redirect? == Think electrophoresis should be seperate article. Electrophoresis refers to the movement of charge particles in a medium induced by an electric potential, and gel electrophoresis is merely the most commom application, strongly recommend that electrophoresis not redirect to this article. Does not adequately explain concepts such as the role of the zeta potential, sediment potentials, streaming potential, electrophoretic mobility etc, nor does it explain related phenomena such as electroosmosis and other electrokinetic actions. Also does not allow for proper understanding of equations modelling electrophoretic movement for example Smoluchowski equation and O'Brien and White's model. Strongly believe electrophoresis should be a more general article linking to this page and other applications such as electrophoretic deposition.
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Gel electrophoresis
Gel electrophoresis is a group of techniques used by scientists to separate molecules based on physical characteristics such as size, shape, or isoelectric point. Gel electrophoresis is usually performed for analytical purposes, but may be used as a preparative technique to partially purify molecules prior to use of other methods such as mass spectrometry, Polymerase chain reaction, cloning, DNA sequencing, or immuno-blotting for further characterization. The first part, "gel", refers to the matrix used to separate the molecules. In most cases the gel is a crosslinked polymer whose porosity can be controlled by the scientist. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually made with different concentrations of acrylamide and a cross-linker, producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a few hundred base (chemistry)), the preferred matrix is purified agarose. In both cases, the gel forms a solid but porous matrix that looks and feels like clear jello. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and needs to be handled using Good Laboratory Practices (GLP) to avoid poisoning. The second part, "electrophoresis", refers to the electromotive force (EMF) that is used to push or pull the molecules through the gel matrix; by placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, towards the anode if negatively charged or towards the cathode if positively charged (note that gel electrophoresis operates as an electrolytic cell; the anode is positive and the cathode is negative). In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the natural negative charge carried on their sugar-phosphate backbone. Double-stranded DNA fragments natually behave as long rods, so their migration through the gel is relative to their radius of gyration, or, roughly, size. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to renature the nucleic acids and cause them to behave as long rods again. Proteins, on the other hand, can have different charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are ususally denatured in the presense of a detergent such as sodium dodecyl sulfate/sodium docecyl phosphate (SDS/SDP) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the size of the protein, so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like they were long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape. After the electrophoresis run, when the smallest molecules have almost reached the anode, the molecules in the gel can be staining (biology) to make them visible. Ethidium bromide, Silver, or Coomassie blue dye can be used. Other methods can also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules luminescence under ultraviolet light, a photograph can be taken of the gel under ultraviolet light. If the molecules to be separated contain radioactivity atoms, an isotopic tracer can be recorded of the gel (as in the example shown above). If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components. Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are special markers available - ladders - which contain a mxiture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule. ==Types== Gel electrophoresis is used in molecular biology, genetics, and biochemistry: *Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis. See the "Chain termination method" page for an example of a Acrylamide DNA sequencing gel. *Gel electrophoresis of proteins is usually done in an sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), by native gel electrophoresis, or by 2-D electrophoresis. *Capillary electrophoresis *DNA electrophoresis *Zymography ==See also== * Protein electrophoresis * Electrofocusing * Southern blotting * Northern blotting * Western blotting ==External links== * [http://gslc.genetics.utah.edu/units/biotech/gel/ Biotechniques Laboratory electrophoresis demonstration], from the University of Utah's Genetic Science Learning Center * [http://www.westernblotting.org Protein electrophoresis and Western Blotting] Molecular biologyBiochemistryAnalytical chemistry Laboratory techniques
Gel electrophoresis
Umm, this is mostly about molecules, not about electrophoresis... -- user:Marj Tiefert == Gel electrophoresis =/ electrophoresis == USP<726> cites Gel, Disk, Zone among others. Also, USP<727> Capillary Electrophoresis, as well as Capillary Zone electrophoresis. Perhaps electrophoresis should not redirect here. Eh, I'm an idiot, although I do still think Gel electrophoresis is still too specific for electrophoresis to redirect here.User:Ix 01:47, 31 May 2004 (UTC) ---- Its not very accurate either. Normally electrophoresis of marcomolecules separates based on size and shape, rather than charge, or at least that's the way it is used. You can also separate based upon the isoelectric point. I think it would be worthwhile redoing the article. This probably should be one place that electrophoresis is linked, but surely not the only one. User:Srlasky 22:43, 2004 Oct 24 (UTC) == Electrophoresis redirect? == Think electrophoresis should be seperate article. Electrophoresis refers to the movement of charge particles in a medium induced by an electric potential, and gel electrophoresis is merely the most commom application, strongly recommend that electrophoresis not redirect to this article. Does not adequately explain concepts such as the role of the zeta potential, sediment potentials, streaming potential, electrophoretic mobility etc, nor does it explain related phenomena such as electroosmosis and other electrokinetic actions. Also does not allow for proper understanding of equations modelling electrophoretic movement for example Smoluchowski equation and O'Brien and White's model. Strongly believe electrophoresis should be a more general article linking to this page and other applications such as electrophoretic deposition.
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