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. Electrophoresis is the most well-known electrokinetic phenomenon. Capillary electrophoresis ( CE) also known as capillary zone electrophoresis (CZE can be used to separate ionic species by their charge and frictional forces SDS-PAGE, Sodium dodecyl sulfate Polyacrylamide gel Electrophoresis, is a technique widely used in Biochemistry, forensics Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of Gel electrophoresis commonly used to analyze proteins Temperature Gradient Gel Electrophoresis ( TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE are forms of Electrophoresis where there is a temperature Deoxyribonucleic acid ( DNA) is a Nucleic acid that contains the genetic instructions used in the development and functioning of all known Ribonucleic acid ( RNA) is a Nucleic acid that consists of a long chain of Nucleotide units Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl In Chemistry, a molecule is defined as a sufficiently stable electrically neutral group of at least two Atoms in a definite arrangement held together by ^[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. Mass spectrometry is an analytical technique that identifies the chemical composition of a compound or sample based on the Mass-to-charge ratio of charged particles A Restriction fragment length polymorphism (or RFLP, often pronounced as "rif-lip" is a variation in the DNA sequence of a Genome Cloning in Biology is the process of producing populations of genetically-identical individuals that occurs in nature when organisms such as Bacteria, Insects The term DNA sequencing encompasses biochemical methods for determining the order of the Nucleotide bases Adenine, Guanine, Cytosine A Southern blot is a method routinely used in Molecular biology to check for the presence of a DNA sequence in a DNA sample

Separation

The term "gel" in this instance refers to the matrix used to contain, then separate the target molecules. A gel (from the lat gelu &mdashfreezing cold ice or gelatus &mdashfrozen immobile is an apparently solid jelly-like material formed from a 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. Cross-links are bonds that link one Polymer chain to another They can be Covalent bonds or Ionic bonds "Polymer chains" can refer 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. Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl A nucleic acid is a Macromolecule composed of chains of monomeric Nucleotides In Biochemistry these Molecules carry Genetic information Deoxyribonucleic acid ( DNA) is a Nucleic acid that contains the genetic instructions used in the development and functioning of all known Ribonucleic acid ( RNA) is a Nucleic acid that consists of a long chain of Nucleotide units An oligonucleotide (or oligo) is a short segment of RNA or DNA, typically with twenty or fewer bases. The Chemical compound acrylamide (acrylic Amide) has the Chemical formula C 3 H 5 N[[Oxygen O]] Cross-links are bonds that link one Polymer chain to another They can be Covalent bonds or Ionic bonds "Polymer chains" can refer Polyacrylamide ( IUPAC poly(2-propenamide or poly(1-carbamoylethylene) is a polymer (-CH2CHCONH2- formed from Acrylamide When separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is purified agarose. In Chemistry, a base is most commonly thought of as an aqueous substance that can accept Protons This refers to the Brønsted-Lowry theory of acids and Agar or agar agar is a Gelatinous substance derived from Seaweed. 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. The Chemical compound acrylamide (acrylic Amide) has the Chemical formula C 3 H 5 N[[Oxygen O]] Polyacrylamide ( IUPAC poly(2-propenamide or poly(1-carbamoylethylene) is a polymer (-CH2CHCONH2- formed from Acrylamide A neurotoxin is a Toxin that acts specifically on nerve cells ( Neurons, usually by interacting with Membrane proteins such as Ion channels Good Laboratory Practice generally refers to a system of management controls for laboratories and research organisations to ensure the consistency and reliability

"Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix. Electrophoresis is the most well-known electrokinetic phenomenon. Electromotive force ( emf, \mathcal{E} is a term used to characterize electrical devices such as Voltaic cells thermoelectric devices electrical 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]. An anode is an Electrode through which Electric current flows into a polarized electrical device A cathode is an Electrode through which (positive Electric current flows out of a polarized electrical device

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.

After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. Staining is an auxiliary Technique used in Microscopy to enhance contrast in the microscopic image Ethidium bromide, silver, or coomassie blue dye may be used for this process. Ethidium bromide (sometimes abbreviated as "EtBr", the abbreviation also confusingly used for Bromoethane) is an intercalating agent Coomassie dyes (also known as Coomassie Brilliant Dyes are a family of dyes commonly used to stain proteins in Sodium dodecyl sulfate and blue native Polyacrylamide 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. Fluorescence is a Luminescence that is mostly found as an Ultraviolet ( UV) light is Electromagnetic radiation with a Wavelength shorter than that of Visible light, but longer than X-rays A photograph (often shortened to photo) is an Image created by Light falling on a light-sensitive surface usually Photographic film or an electronic If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel. Radioactive decay is the process in which an unstable Atomic nucleus loses energy by emitting ionizing particles and Radiation. Isotopic labeling is a technique for tracking the passage of a sample of substance through a system

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. A molecular weight size marker is used to identify the approximate size of a molecule run on a Gel electrophoresis. 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.

Applications

Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemistry. 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 Genetics (from Ancient Greek grc-Latn genetikos, “genitive” and that from grc-Latn genesis, “origin” a discipline of Biology, is Microbiology (from Greek grc μῑκρος mīkros, "small" grc βίος bios, " Life " and grc -λογία Biochemistry is the study of the chemical processes in living Organisms It deals with the Structure and function of cellular components such as 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.

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. Sugar is a class of edible Crystalline substances mainly Sucrose, Lactose, and Fructose. A phosphate, an Inorganic chemical, is a salt of Phosphoric acid. ^[3]

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. Radius of gyration is the name of several related measures of the size of an object a surface or an ensemble of points 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. A hydrogen bond results from a Dipole-dipole force between an Electronegative atom and a Hydrogen atom bonded to Nitrogen, Oxygen Sodium hydroxide ( Na[[hydroxide OH]]) also known as Lye, caustic soda and (incorrectly according to IUPAC nomenclature Formamide, also known as methanamide is an Amide derived from Formic acid. ^[4]

Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis. Deoxyribonucleic acid ( DNA) is a Nucleic acid that contains the genetic instructions used in the development and functioning of all known Ribonucleic acid ( RNA) is a Nucleic acid that consists of a long chain of Nucleotide units Agarose Gel electrophoresis is a method used in Biochemistry and Molecular biology to separate DNA, or RNA molecules by size See the "Chain termination method" page for an example of a polyacrylamide DNA sequencing gel. The term DNA sequencing encompasses biochemical methods for determining the order of the Nucleotide bases Adenine, Guanine, Cytosine Polyacrylamide ( IUPAC poly(2-propenamide or poly(1-carbamoylethylene) is a polymer (-CH2CHCONH2- formed from Acrylamide

Proteins

SDS-PAGE autoradiography - The indicated proteins are present in different concentrations in the two samples. An autoradiograph is an image on an X-ray film or nuclear emulsion produced by the pattern of decay emissions (e

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. Denaturation is a process in which Proteins or Nucleic acids lose their structure (tertiary structure by application of some external stress or compound for A detergent (as a noun is a material intended to assist Cleaning. Sodium lauryl sulfate ( SLS) or sodium dodecyl sulfate ( SDS or NaDS ( C 12 H 25 S[[oxygen O]]4 ^[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]

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. Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl SDS-PAGE, Sodium dodecyl sulfate Polyacrylamide gel Electrophoresis, is a technique widely used in Biochemistry, forensics Native Gel Electrophoresis is a technique used mainly in Protein electrophoresis where the proteins are not denatured and therefore separated based on their charge-to-mass ratio QPNC-PAGE, or quantitative preparative native continuous polyacrylamide gel electrophoresis, is a high-resolution technique applied in Biochemistry and Bioinorganic Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of Gel electrophoresis commonly used to analyze proteins

History

• 1930s - first reports of the use of sucrose for gel electrophoresis
• 1955 - introduction of starch gels, mediocre separation
• 1959 - introduction of acrylamide gels (Raymond and Weintraub); accurate control of parameters such as pore size and stability
• 1964 - disc gel electrophoresis (Ornstein and Davis)
• 1969 - introduction of denaturing agents especially SDS separation of protein subunit (Beber and Osborn)
• 1970 - Laemmli separated 28 components of T4 phage using a stacking gel and SDS
• 1975 - 2-dimensional gels (O’Farrell); isoelectric focusing then SDS gel electrophoresis
• 1977 - sequencing gels
• late 1970s - agarose gels
• 1983 - pulsed field gel electrophoresis enables separation of large DNA moleucles
• 1983 - introduction of capillary electrophoresis

A 1959 book on electrophoresis by Milan Bier cites references from the 1800s. 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. Solubility of Pure SucroseTemperature(Cg Year 1955 ( MCMLV) was a Common year starting on Saturday (link displays the 1955 Gregorian calendar) Starch, CAS # 9005-25-8 Chemical formula (C6H10O5n is a Polysaccharide The year 1959 ( MCMLIX) was a Common year starting on Thursday (link will display full calendar of the Gregorian calendar. The Chemical compound acrylamide (acrylic Amide) has the Chemical formula C 3 H 5 N[[Oxygen O]] Year 1964 ( MCMLXIV) was a Leap year starting on Wednesday (link will display full calendar of the 1964 Gregorian calendar. Year 1969 ( MCMLXIX) was a Common year starting on Wednesday (link will display full calendar of the Gregorian calendar. Denaturation is a process in which Proteins or Nucleic acids lose their structure (tertiary structure by application of some external stress or compound for Sodium lauryl sulfate ( SLS) or sodium dodecyl sulfate ( SDS or NaDS ( C 12 H 25 S[[oxygen O]]4 Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl 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 (IEF, also known as electrofocusing, is a technique for separating different Molecules by their Electric charge differences Also 1977 (album by Ash. Year 1977 ( MCMLXXVII) was a Common year starting on Saturday (link displays The term DNA sequencing encompasses biochemical methods for determining the order of the Nucleotide bases Adenine, Guanine, Cytosine This article is about the Decade 1970-1979 For the Year 1970 see 1970. Agar or agar agar is a Gelatinous substance derived from Seaweed. Year 1983 ( MCMLXXXIII) was a Common year starting on Saturday (link displays the 1983 Gregorian calendar) Historical Background Standard gel electrophoresis techniques for separation of DNA molecules provided huge advantages for molecular biology research 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 ^[5] However, Oliver Smithies made significant contributions. Oliver Smithies (born June 23 1925) is a British-born American geneticist and Nobel laureate, credited with the invention 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.

See also

• DNA electrophoresis
• Electrofocusing
• Gel isolation
• Northern blotting
• Protein electrophoresis
• Southern blotting
• Western blotting
• Zymography
• Native Gel Electrophoresis

References

External links

• Biotechniques Laboratory electrophoresis demonstration, from the University of Utah's Genetic Science Learning Center
• Discontinuous native protein gel electrophoresis