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Encyclopedia > Gel electrophoresis
Gel electrophoresis

Gel electrophoresis apparatus - An agarose gel is placed in this buffer-filled box and electrical current is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates toward the camera.
Classification Electrophoresis
Other Techniques
Related Capillary electrophoresis
SDS-PAGE
Two-dimensional gel electrophoresis
Temperature gradient gel electrophoresis
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Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid, ribonucleic acid, or protein molecules using an electric current applied to a gel matrix.[1] It is usually performed for analytical purposes, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization. Image File history File linksMetadata Download high resolution version (1476x1886, 598 KB) File links The following pages link to this file: Gel electrophoresis Portal:Biotechnology/Title Template:Biotechnology title 55 Metadata This file contains additional information, probably added from the digital camera or scanner used to create or digitize it. ... For specific types of electrophoresis (for example, the process of administering medicine, iontophoresis), see electrophoresis (disambiguation). ... Capillary electrophoresis (CE), also known as capillary zone electrophoresis (CZE), can be used to separate ionic species by their charge and frictional forces. ... Picture of an SDS-PAGE. The molecular marker is in the left lane SDS-PAGE stands for Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. ... Two-dimensional gel electrophoresis, commonly abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. ... Temperature gradient gel electrophoresis (TGGE) is a form of electrophoresis that studies the behavior of substances under different temperatures. ... The structure of part of a DNA double helix Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. ... For other uses, see RNA (disambiguation). ... A representation of the 3D structure of myoglobin showing coloured alpha helices. ... 3D (left and center) and 2D (right) representations of the terpenoid molecule atisane. ... Mass spectrometry (previously called mass spectroscopy (deprecated) or informally, mass-spec and MS) is an analytical technique that measures the mass-to-charge ratio of ions. ... In molecular biology, the term restriction fragment length polymorphism (or RFLP, often pronounced rif-lip) is used in two related contexts: as a characteristic of DNA molecules (arising from their differing nucleotide sequences) by which they may be distinguished and as the laboratory technique which uses this characteristic to compare... “PCR” redirects here. ... For the cloning of human beings, see human cloning. ... The term DNA sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. ... A Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. ...

Contents

Separation

The term "gel" in this instance refers to the matrix used to contain, then separate the target molecules. In most cases the gel is a crosslinked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually composed of 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 bases), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be handled using Good Laboratory Practices to avoid poisoning. In optical filters and theatrical lighting a color gel is a transparent or translucent colored panel used to change the color of transmitted light. ... Vulcanization is an example of cross-linking. ... A representation of the 3D structure of myoglobin showing coloured alpha helices. ... Look up nucleic acid in Wiktionary, the free dictionary. ... The structure of part of a DNA double helix Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. ... For other uses, see RNA (disambiguation). ... Oligonucleotides are short sequences of nucleotides (RNA or DNA), typically with twenty or fewer bases. ... R-phrases , , , , , , , S-phrases , Flash point 138 °C Autoignition temperature 424 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references The chemical compound acrylamide (acrylic amide) has the chemical formula C3H5NO. Its IUPAC name is 2... Vulcanization is an example of cross-linking. ... Polyacrylamide is an acrylate polymer formed from acrylamide subunits that is readily cross-linked. ... Acids and bases: Acid-base extraction Acid-base reaction Acid dissociation constant Acidity function Buffer solutions pH Proton affinity Self-ionization of water Acids: Lewis acids Mineral acids Organic acids Strong acids Superacids Weak acids Bases: Lewis bases Organic bases Strong bases Superbases Non-nucleophilic bases Weak bases edit In... An agarose is a polysaccharide polymer material, generally extracted from seaweed. ... R-phrases , , , , , , , S-phrases , Flash point 138 °C Autoignition temperature 424 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references The chemical compound acrylamide (acrylic amide) has the chemical formula C3H5NO. Its IUPAC name is 2... Polyacrylamide is an acrylate polymer formed from acrylamide subunits that is readily cross-linked. ... A neurotoxin is a toxin that acts specifically on nerve cells – neurons – usually by interacting with membrane proteins such as ion channels. ... GLP It is important to be safe when playing in a lab. ...


"Electrophoresis" refers to the electromotive force (EMF) that is used to move 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, usually determined by mass, toward the positive anode if negatively charged or toward the negative cathode if positively charged [2]. For specific types of electrophoresis (for example, the process of administering medicine, iontophoresis), see electrophoresis (disambiguation). ... Electromotive force (emf) is the amount of energy gained per unit charge that passes through a device in the opposite direction to the electric field existing across that device. ... Diagram of a zinc anode in a galvanic cell. ... Diagram of a copper cathode in a Daniells cell. ...


Visualization

I Agarose gel prepared for DNA analysis - The first lane contains a DNA ladder for sizing, and the other four lanes show variously-sized DNA fragments that are present in some but not all of the samples.
I Agarose gel prepared for DNA analysis - The first lane contains a DNA ladder for sizing, and the other four lanes show variously-sized DNA fragments that are present in some but not all of the samples.

After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie blue dye may be used for this process. Other methods may also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel. Example of DNA agarose gel electrophoresis. ... Example of DNA agarose gel electrophoresis. ... Staining is a biochemical technique of adding a class-specific (DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound. ... R-phrases , S-phrases , , , , , Flash point > 100 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references Absorption spectrum of ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic... Solution of Coomassie Coomassie (also known as Coomassie Blue, Brilliant Blue, Brilliant Blue G, Acid Blue 90, C.I. 42655, Brilliant Blue G 250, or Kunasty Blue) is a blue dye commonly used in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. ... For other uses, see Ultraviolet (disambiguation). ... For other uses, see Photograph (disambiguation). ... Radioactivity may mean: Look up radioactivity in Wiktionary, the free dictionary. ... An isotopic tracer, (also isotopic marker or isotopic label), is used in chemistry and biochemistry to help understand chemical reactions and interactions. ...


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 molecular weight size markers available that contain a mixture 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. A molecular weight size marker is used to identify the approximate size of a molecule run on a gel electrophoresis. ...


Applications

Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemistry. The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device. The image is recorded with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel. The measurement and analysis are mostly done with specialized software. Forensic chemistry is the application of chemistry to law enforcement or the failure of products or processes. ... Molecular biology is the study of biology at a molecular level. ... This article is about the general scientific term. ... An agar plate streaked with microorganisms Microbiology is the study of microorganisms, which are unicellular or cell-cluster microscopic organisms. ... Biochemistry (from Greek: , bios, life and Egyptian kēme, earth[1]) is the study of the chemical processes in living organisms. ...


Depending on the type of analysis being performed, other techniques are often implemented in conjunction with the results of gel electrophoresis, providing a wide range of field-specific applications.


Nucleic acids

In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the naturally-occurring negative charge carried by their sugar-phosphate backbone.[3] This article is about sugar as food and as an important and widely-traded commodity. ... A phosphate, in inorganic chemistry, is a salt of phosphoric acid. ...


Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their radius of gyration, or, for non-cyclic fragments, 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 denature the nucleic acids and cause them to behave as long rods again.[4] The radius of gyration is a defined measure for the dimension of an object, a surface, or an ensemble of points. ... An example of a quadruple hydrogen bond between a self-assembled dimer complex reported by Meijer and coworkers. ... Sodium hydroxide (NaOH), also known as lye, caustic soda and (incorrectly, according to IUPAC nomenclature)[1] sodium hydrate, is a caustic metallic base. ... Formamide, also known as Methanamide (IUPAC) and Carbamaldehyde, formula HCONH2 is an amide derived from formic acid. ...


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 polyacrylamide DNA sequencing gel. The structure of part of a DNA double helix Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. ... For other uses, see RNA (disambiguation). ... Digital image of 3 plasmid restriction digests run on a 1% w/v agarose gel, 3 Volts/cm, stained with ethidium bromide. ... The chain termination or Sanger or dideoxy method is a process used to sequence (read the bases of) DNA. It is named after Frederick Sanger who developed the process in 1975. ... R-phrases , , , , , , , S-phrases , Flash point 138 °C Autoignition temperature 424 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references The chemical compound acrylamide (acrylic amide) has the chemical formula C3H5NO. Its IUPAC name is 2...


Proteins

SDS-PAGE autoradiography - The indicated proteins are present in different concentrations in the two samples.
SDS-PAGE autoradiography - The indicated proteins are present in different concentrations in the two samples.

Proteins, unlike nucleic acids, can have varying 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 usually denatured in the presence of a detergent such as sodium dodecyl sulfate/sodium dodecyl phosphate (SDS/SDP) that coats the proteins with a negative charge.[1] Generally, the amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per gram of 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 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.[1] Example of SDS-PAGE of proteins visualized by autoradiography. ... Example of SDS-PAGE of proteins visualized by autoradiography. ... An autoradiograph is an image produced on a photographic film by the radiation from a radioactive substance. ... Irreversible egg protein denaturation and loss of solubility, caused by the high temperature (while cooking it) Denaturation is the alteration of a protein or nucleic acids shape through some form of external stress (for example, by applying heat, acid or alkali), in such a way that it will no... Laundry detergents are just one of many possible uses for detergents Detergent is a compound, or a mixture of compounds, intended to assist cleaning. ... Sodium dodecyl sulfate (or sulphate) (SDS or NaDS) (C12H25NaO4S), also known as sodium lauryl sulfate (SLS), is an ionic surfactant that is used in household products such as toothpastes, shampoos, shaving foams and bubble baths for its thickening effect and its ability to create a lather. ...


Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), by native gel electrophoresis, by quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE), or by 2-D electrophoresis. A representation of the 3D structure of myoglobin showing coloured alpha helices. ... Picture of an SDS-PAGE. The molecular marker is in the left lane SDS-PAGE stands for Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. ... QPNC-PAGE (Abbr. ... Two dimensional gel electrophoresis, commonly abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. ...


History

A 1959 book on electrophoresis by Milan Bier cites references from the 1800s.[5] However, Oliver Smithies made significant contributions. Bier states: "The method of Smithies ... is finding wide application because of its unique separatory power." Taken in context, Bier clearly implies that Smithies' method is an improvement. The 1930s were described as an abrupt shift to more radical and conservative lifestyles, as countries were struggling to find a solution to the Great Depression, also known as the [[. In East Asia, the rise of militarism occurred. ... Flash point N/A Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references Sucrose (common name: table sugar, also called saccharose) is a disaccharide (glucose + fructose) with the molecular formula C12H22O11. ... Year 1955 (MCMLV) was a common year starting on Saturday (link displays the 1955 Gregorian calendar). ... Starch (CAS# 9005-25-8, chemical formula (C6H10O5)n,[1]) is a mixture of amylose and amylopectin (usually in 20:80 or 30:70 ratios). ... Year 1959 (MCMLIX) was a common year starting on Thursday (link will display full calendar) of the Gregorian calendar. ... R-phrases , , , , , , , S-phrases , Flash point 138 °C Autoignition temperature 424 °C Except where noted otherwise, data are given for materials in their standard state (at 25 Â°C, 100 kPa) Infobox disclaimer and references The chemical compound acrylamide (acrylic amide) has the chemical formula C3H5NO. Its IUPAC name is 2... Also Nintendo emulator: 1964 (emulator). ... Also: 1969 (number) 1969 (movie) 1969 (Stargate SG-1) episode. ... Irreversible egg protein denaturation and loss of solubility, caused by the high temperature (while cooking it) Denaturation is the alteration of a protein or nucleic acids shape through some form of external stress (for example, by applying heat, acid or alkali), in such a way that it will no... Sodium dodecyl sulfate (or sulphate) (SDS or NaDS) (C12H25NaO4S), also known as sodium lauryl sulfate (SLS), is an ionic surfactant that is used in household products such as toothpastes, shampoos, shaving foams and bubble baths for its thickening effect and its ability to create a lather. ... A representation of the 3D structure of myoglobin showing coloured alpha helices. ... Year 1970 (MCMLXX) was a common year starting on Thursday (link shows full calendar) of the Gregorian calendar. ... Enterobacteria phage T4 is a phage that infects E. coli bacteria. ... Year 1975 (MCMLXXV) was a common year starting on Wednesday (link will display full calendar) of the Gregorian calendar. ... Isoelectric focusing, also known as electrofocusing, is a technique for separating different molecules by their electric charge differences. ... Also: 1977 (album) by Ash. ... The term DNA sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. ... The 1970s decade refers to the years from 1970 to 1979, also called The Seventies. ... An agarose is a polysaccharide polymer material, generally extracted from seaweed. ... Year 1983 (MCMLXXXIII) was a common year starting on Saturday (link displays the 1983 Gregorian calendar). ... Pulsed Field Gel Electrophoresis (commonly abbreviated as PFGE) is a labor-intensive method for genetic fingerprinting. ... Year 1983 (MCMLXXXIII) was a common year starting on Saturday (link displays the 1983 Gregorian calendar). ... Capillary electrophoresis (CE), also known as capillary zone electrophoresis (CZE), can be used to separate ionic species by their charge and frictional forces. ... // Invention of the Jacquard loom in 1801. ... Oliver Smithies (born July 23, 1925) is a British-born American geneticist and Nobel laureate,[1] credited with the discovery of gel electrophoresis in 1950, and the simultaneous discovery, with Mario Capecchi, of the technique of homologous recombination of transgenic DNA with genomic DNA, a much more reliable method of...


See also

Digital printout of an agarose gel electrophoresis of cat-insert plasmid DNA DNA electrophoresis is an analytical technique used to separate DNA fragments by size. ... Electrofocusing, or isoelectric focusing, is a technique for separating different molecules by their electric charge differences (if they have any charge). ... In molecular biology, gel extraction or gel isolation is a technique where viable DNA is eluted (extracted) from an agarose gel following agarose gel electrophoresis in order to isolate a specific band. ... The northern blot is a technique used in molecular biology research to study gene expression. ... Schematic representation of a protein electrophoresis gel In chemistry and medicine, protein electrophoresis (a. ... A Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. ... A Western blot. ... Zymography is an electrophoretic technique, based on SDS-PAGE, that includes a substrate copolymerised with the polyacrylamide gel, for the detection of enzyme activity. ...

References

  1. ^ a b c Berg JM, Tymoczko JL Stryer L (2002). Molecular Cell Biology, 5th ed., WH Freeman. ISBN 0-7167-4955-6. 
  2. ^ Robyt, John F. (1990). Biochemical Techniques Theory and Practice. Waveland Press. ISBN 0-88133-556-8. 
  3. ^ Lodish H, Berk A, Matsudaira P, et al (2004). Molecular Cell Biology, 5th ed., WH Freeman: New York, NY. ISBN 978-0716743668. 
  4. ^ Troubleshooting DNA agarose gel electrophoresis. Focus 19:3 p.66 (1997).
  5. ^ Milan Bier (ed.) (1959). Electrophoresis. Theory, Methods and Applications, 3rd printing, Academic Press, 225. LCC 59-7676. OCLC 1175404. 

The Online Computer Library Center (OCLC) was founded in 1967 and originally named the Ohio College Library Center. ...

External links

  • Biotechniques Laboratory electrophoresis demonstration, from the University of Utah's Genetic Science Learning Center
  • Discontinuous native protein gel electrophoresis
A representation of the 3D structure of myoglobin showing coloured alpha helices. ... Protein methods are the techniques used to study proteins. ... In the scientific method, an experiment (Latin: ex- periri, of (or from) trying) is a set of observations performed in the context of solving a particular problem or question, to retain or falsify a hypothesis or research concerning phenomena. ... -1... It has been suggested that mGFP be merged into this article or section. ... A Western blot. ... Immunostaining is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. ... Proteins are found in every cell and are essential to every biological process, protein structure is very complex: determining a proteins structure involves first protein sequencing - determining the amino acid sequences of its constituent peptides; and also determining what conformation it adopts and whether it is complexed with any... Schematic representation of a protein electrophoresis gel In chemistry and medicine, protein electrophoresis (a. ... Immunoprecipitation is the technique of precipitating an antigen out of solution using an antibody specific to that antigen. ... Peptide mass fingerprinting (also known as protein fingerprinting) (PMF) is an analytical technique for protein identification that was developed by John Yates and colleagues (3). ... Map of the human X chromosome (from the NCBI website). ... Protein structure prediction is one of the most significant technologies pursued by computational structural biology and theoretical chemistry. ... Protein-protein docking is the determination of the molecular structure of complexes formed by two or more proteins without the need for experimental measurement. ... Protein structural alignment is a form of alignment which tries to establish equivalences between two or more protein structures based on their fold. ... Protein ontology or Proteome Ontology is a research tool of proteomics, similar to the scientific classification system used in biology. ... This article is being considered for deletion in accordance with Wikipedias deletion policy. ... An assay is a procedure where the concentration of a component part of a mixture is determined. ... Enzyme assays are laboratory methods for measuring enzymatic activity. ... The Bradford Protein Assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. ... Secretion assay is a process used in cell biology to identify cells that are secreting a particular protein (usually a cytokine). ...

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