{"history":[],"article":{"id":"d840dfbc-0580-4706-9025-5605437af61a","title":"Gel electrophoresis","slug":"gel-electrophoresis","content":":::summary\n![Gel electrophoresis apparatus – an agarose gel is placed in this buffer-filled box and an electric 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 positively charged anode(red wire). This occurs because phosphate groups found in the DNA fragments possess a negative charge which is repelled by the negatively charged cathode and are attracted to the positively charged anode.](https://upload.wikimedia.org/wikipedia/commons/a/a6/Gel///electrophoresis///apparatus.JPG)\n**Name:** Gel electrophoresis \n**Classification:** [Electrophoresis](https://bliptext.com/articles/electrophoresis) \n**Related:** [Capillary electrophoresis](https://bliptext.com/articles/capillary-electrophoresis) [SDS-PAGE](https://bliptext.com/articles/polyacrylamide-gel-electrophoresis) [Two-dimensional gel electrophoresis](https://bliptext.com/articles/two-dimensional-gel-electrophoresis) [Temperature gradient gel electrophoresis](https://bliptext.com/articles/temperature-gradient-gel-electrophoresis) \n:::\n\n# \n**Gel electrophoresis** is a method for separation and analysis of [biomacromolecules](https://bliptext.com/articles/biomolecule) ([DNA](https://bliptext.com/articles/dna), [RNA](https://bliptext.com/articles/rna), [proteins](https://bliptext.com/articles/protein), etc.) and their fragments, based on their size and charge.\nIt is used in [clinical chemistry](https://bliptext.com/articles/clinical-chemistry) to separate proteins by charge or size (IEF agarose, essentially size independent) and in [biochemistry](https://bliptext.com/articles/biochemistry) and [molecular biology](https://bliptext.com/articles/molecular-biology) to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.\nNucleic acid molecules are separated by applying an [electric field](https://bliptext.com/articles/electric-field) to move the negatively charged molecules through a matrix of [agarose](https://bliptext.com/articles/agarose) or other substances.\nShorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel.\nThis phenomenon is called sieving.\nProteins are separated by the charge in agarose because the pores of the gel are too large to sieve proteins.\nGel electrophoresis can also be used for the separation of [nanoparticles](https://bliptext.com/articles/nanoparticle).\nGel electrophoresis uses a gel as an anticonvective medium or sieving medium during electrophoresis, the movement of a charged particle in an electric current.\nGels suppress the thermal convection caused by the application of the electric field, and can also act as a sieving medium, slowing the passage of molecules; gels can also simply serve to maintain the finished separation so that a post electrophoresis stain can be applied.\nDNA gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via [polymerase chain reaction](https://bliptext.com/articles/polymerase-chain-reaction) (PCR), but may be used as a preparative technique prior to use of other methods such as [mass spectrometry](https://bliptext.com/articles/mass-spectrometry), [RFLP](https://bliptext.com/articles/restriction-fragment-length-polymorphism), PCR, [cloning](https://bliptext.com/articles/cloning), [DNA sequencing](https://bliptext.com/articles/dna-sequencing), or [Southern blotting](https://bliptext.com/articles/southern-blot) for further characterization.\n# [The image above shows how small DNA fragments will migrate through agarose quickly but large size DNA fragments move more slowly during electrophoresis. The graph to the right shows the nonlinear relationship between the size of the DNA fragment and the distance migrated. | upload.wikimedia.org/wikipedia/commons/7/75////Gel///Electrophoresis.svg]\n# [Gel electrophoresis is a process where an electric current is applied to DNA samples creating fragments that can be used for comparison between DNA samples.\n

DNA is extracted.
Isolation and amplification of DNA.
DNA added to the gel wells.
Electric current applied to the gel.
DNA bands are separated by size.
DNA bands are stained.

| upload.wikimedia.org/wikipedia/commons/d/d0/Gel///Electrophoresis///in///DNA///Fingerprinting.svg]\n\n# Physical basis\n[Electrophoresis](https://bliptext.com/articles/electrophoresis) is a process that enables the sorting of molecules based on charge, size, or shape.\nUsing an electric field, molecules (such as DNA) can be made to move through a gel made of [agarose](https://bliptext.com/articles/agarose) or [polyacrylamide](https://bliptext.com/articles/polyacrylamide).\nThe electric field consists of a negative charge at one end which pushes the molecules through the gel, and a positive charge at the other end that pulls the molecules through the gel.\nThe molecules being sorted are dispensed into a well in the gel material.\nThe gel is placed in an electrophoresis chamber, which is then connected to a power source.\nWhen the electric field is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster.\nThe different sized molecules form distinct bands on the gel.\nThe term \"[gel](https://bliptext.com/articles/gel)\" in this instance refers to the matrix used to contain, then separate the target molecules.\nIn most cases, the gel is a [crosslinked polymer](https://bliptext.com/articles/crosslinked-polymer) whose composition and porosity are chosen based on the specific weight and composition of the target to be analyzed.\nWhen separating [proteins](https://bliptext.com/articles/protein) or small [nucleic acids](https://bliptext.com/articles/nucleic-acid) ([DNA](https://bliptext.com/articles/dna), [RNA](https://bliptext.com/articles/rna), or [oligonucleotides](https://bliptext.com/articles/oligonucleotide)) the gel is usually composed of different concentrations of [acrylamide](https://bliptext.com/articles/acrylamide) and a [cross-linker](https://bliptext.com/articles/cross-linker), producing different sized mesh networks of poly[acrylamide](https://bliptext.com/articles/acrylamide).\nWhen separating larger nucleic acids (greater than a few hundred [bases](https://bliptext.com/articles/base-chemistry)), the preferred matrix is purified agarose.\nIn both cases, the gel forms a solid, yet porous matrix.\nAcrylamide, in contrast to polyacrylamide, is a [neurotoxin](https://bliptext.com/articles/neurotoxin) and must be handled using appropriate safety precautions to avoid poisoning.\nAgarose is composed of long unbranched chains of uncharged carbohydrates without cross-links resulting in a gel with large pores allowing for the separation of macromolecules and [macromolecular complexes](https://bliptext.com/articles/affinity-electrophoresis).\nElectrophoresis refers to the [electromotive force](https://bliptext.com/articles/electromotive-force) (EMF) that is used to move the molecules through the gel matrix.\nBy placing the molecules in wells in the gel and applying an electric field, the molecules will move through the matrix at different rates, determined largely by their mass when the charge-to-mass ratio (Z) of all species is uniform.\nHowever, when charges are not all uniform the electrical field generated by the electrophoresis procedure will cause the molecules to migrate differentially according to charge.\nSpecies that are net positively charged will migrate towards the [cathode](https://bliptext.com/articles/cathode) which is negatively charged (because this is an [electrolytic](https://bliptext.com/articles/electrolytic-cell) rather than [galvanic cell](https://bliptext.com/articles/galvanic-cell)), whereas species that are net negatively charged will migrate towards the positively charged anode.\nMass remains a factor in the speed with which these non-uniformly charged molecules migrate through the matrix toward their respective electrodes.\nIf several samples have been loaded into adjacent wells in the gel, they will run parallel in individual lanes.\nDepending on the number of different molecules, each lane shows the separation of the components from the original mixture as one or more distinct bands, one band per component.\nIncomplete separation of the components can lead to overlapping bands, or indistinguishable smears representing multiple unresolved components.\nBands in different lanes that end up at the same distance from the top contain molecules that passed through the gel at the same speed, which usually means they are approximately the same size.\nThere are [molecular weight size markers](https://bliptext.com/articles/molecular-weight-size-marker) available that contain a mixture of molecules of known sizes.\nIf 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 to determine their size.\nThe distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule (alternatively, this can be stated as the distance traveled is inversely proportional to the log of samples's molecular weight).\nThere are limits to electrophoretic techniques.\nSince passing a current through a gel causes heating, gels may melt during electrophoresis.\nElectrophoresis is performed in buffer solutions to reduce pH changes due to the electric field, which is important because the charge of DNA and RNA depends on pH, but running for too long can exhaust the buffering capacity of the solution.\nThere are also limitations in determining the molecular weight by SDS-PAGE, especially when trying to find the MW of an unknown protein.\nCertain biological variables are difficult or impossible to minimize and can affect electrophoretic migration.\nSuch factors include protein structure, post-translational modifications, and amino acid composition.\nFor example, tropomyosin is an acidic protein that migrates abnormally on SDS-PAGE gels.\nThis is because the acidic residues are repelled by the negatively charged SDS, leading to an inaccurate mass-to-charge ratio and migration.\nFurther, different preparations of genetic material may not migrate consistently with each other, for morphological or other reasons.\n# [Overview of gel electrophoresis. | upload.wikimedia.org/wikipedia/commons/4/46/SDS-PAGE///Electrophoresis.png]\n\n# Types of gel\nThe types of gel most typically used are agarose and polyacrylamide gels.\nEach type of gel is well-suited to different types and sizes of the analyte.\nPolyacrylamide gels are usually used for proteins and have very high resolving power for small fragments of DNA (5-500 bp).\nAgarose gels, on the other hand, have lower resolving power for DNA but have a greater range of separation, and are therefore used for DNA fragments of usually 50–20,000 bp in size, but the resolution of over 6 Mb is possible with [pulsed field gel electrophoresis](https://bliptext.com/articles/pulsed-field-gel-electrophoresis) (PFGE).\nPolyacrylamide gels are run in a vertical configuration while agarose gels are typically run horizontally in a submarine mode.\nThey also differ in their casting methodology, as agarose sets thermally, while polyacrylamide forms in a chemical polymerization reaction.\n\n## Agarose\nAgarose gels are made from the natural [polysaccharide](https://bliptext.com/articles/polysaccharide) [polymers](https://bliptext.com/articles/polymer) extracted from [seaweed](https://bliptext.com/articles/seaweed).\nAgarose gels are easily cast and handled compared to other matrices because the gel setting is a physical rather than chemical change.\nSamples are also easily recovered.\nAfter the experiment is finished, the resulting gel can be stored in a plastic bag in a refrigerator.\nAgarose gels do not have a uniform pore size, but are optimal for electrophoresis of proteins that are larger than 200 kDa.\nAgarose gel electrophoresis can also be used for the separation of DNA fragments ranging from 50 [base pair](https://bliptext.com/articles/base-pair) to several megabases (millions of bases), the largest of which require specialized apparatus.\nThe distance between DNA bands of different lengths is influenced by the percent agarose in the gel, with higher percentages requiring longer run times, sometimes days.\nInstead high percentage agarose gels should be run with a [pulsed field electrophoresis](https://bliptext.com/articles/pulsed-field-gel-electrophoresis) (PFE), or [field inversion electrophoresis](https://bliptext.com/articles/field-inversion-electrophoresis).\n\"Most agarose gels are made with between 0.7% (good separation or resolution of large 5–10kb DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose dissolved in electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1% gels are common for many applications.\"\n# [Inserting the gel comb in an agarose gel electrophoresis chamber | upload.wikimedia.org/wikipedia/commons/5/5b/Gel///electrophoresis///insert///comb.jpg]\n\n## Polyacrylamide\nPolyacrylamide gel electrophoresis (PAGE) is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel.\nPore size is controlled by modulating the concentrations of acrylamide and bis-acrylamide powder used in creating a gel.\nCare must be used when creating this type of gel, as acrylamide is a potent neurotoxin in its liquid and powdered forms.\nTraditional [DNA sequencing](https://bliptext.com/articles/dna-sequencing) techniques such as [Maxam-Gilbert](https://bliptext.com/articles/maxam-gilbert-sequencing) or [Sanger](https://bliptext.com/articles/dideoxy-termination) methods used polyacrylamide gels to separate DNA fragments differing by a single base-pair in length so the sequence could be read.\nMost modern DNA separation methods now use agarose gels, except for particularly small DNA fragments.\nIt is currently most often used in the field of [immunology](https://bliptext.com/articles/immunology) and protein analysis, often used to separate different proteins or [isoforms](https://bliptext.com/articles/isoforms) of the same protein into separate bands.\nThese can be transferred onto a [nitrocellulose](https://bliptext.com/articles/nitrocellulose) or [PVDF](https://bliptext.com/articles/pvdf) membrane to be probed with antibodies and corresponding markers, such as in a [western blot](https://bliptext.com/articles/western-blot).\nTypically [resolving gels](https://bliptext.com/articles/resolving-gels) are made in 6%, 8%, 10%, 12% or 15%.\nStacking gel (5%) is poured on top of the resolving gel and a gel comb (which forms the wells and defines the lanes where proteins, sample buffer, and ladders will be placed) is inserted.\nThe percentage chosen depends on the size of the protein that one wishes to identify or probe in the sample.\nThe smaller the known weight, the higher the percentage that should be used.\nChanges in the buffer system of the gel can help to further resolve proteins of very small sizes.\n\n## Starch\nPartially [hydrolysed](https://bliptext.com/articles/hydrolysis) potato starch makes for another non-toxic medium for protein electrophoresis.\nThe gels are slightly more opaque than acrylamide or agarose.\nNon-denatured proteins can be separated according to charge and size.\nThey are visualised using Napthal Black or Amido Black staining.\nTypical starch gel concentrations are 5% to 10%.\n\n# Gel conditions\n\n## Denaturing\n[Denaturing](https://bliptext.com/articles/denaturation-biochemistry) gels are run under conditions that disrupt the natural structure of the analyte, causing it to unfold into a linear chain.\nThus, the mobility of each [macromolecule](https://bliptext.com/articles/macromolecule) depends only on its linear length and its mass-to-charge ratio.\nThus, the secondary, tertiary, and quaternary levels of [biomolecular structure](https://bliptext.com/articles/biomolecular-structure) are disrupted, leaving only the primary structure to be analyzed.\nNucleic acids are often denatured by including [urea](https://bliptext.com/articles/urea) in the buffer, while proteins are denatured using [sodium dodecyl sulfate](https://bliptext.com/articles/sodium-dodecyl-sulfate), usually as part of the [SDS-PAGE](https://bliptext.com/articles/polyacrylamide-gel-electrophoresis) process.\nFor full denaturation of proteins, it is also necessary to reduce the covalent [disulfide bonds](https://bliptext.com/articles/disulfide-bond) that stabilize their [tertiary](https://bliptext.com/articles/tertiary-structure) and [quaternary structure](https://bliptext.com/articles/quaternary-structure), a method called reducing PAGE.\nReducing conditions are usually maintained by the addition of [beta-mercaptoethanol](https://bliptext.com/articles/beta-mercaptoethanol) or [dithiothreitol](https://bliptext.com/articles/dithiothreitol).\nFor a general analysis of protein samples, reducing PAGE is the most common form of [protein electrophoresis](https://bliptext.com/articles/protein-electrophoresis).\nDenaturing conditions are necessary for proper estimation of molecular weight of RNA.\nRNA is able to form more intramolecular interactions than DNA which may result in change of its [electrophoretic mobility](https://bliptext.com/articles/electrophoretic-mobility).\n[Urea](https://bliptext.com/articles/urea), [DMSO](https://bliptext.com/articles/dimethyl-sulfoxide) and [glyoxal](https://bliptext.com/articles/glyoxal) are the most often used denaturing agents to disrupt RNA structure.\nOriginally, highly toxic [methylmercury](https://bliptext.com/articles/methylmercury) hydroxide was often used in denaturing RNA electrophoresis, but it may be method of choice for some samples.\nDenaturing gel electrophoresis is used in the DNA and RNA banding pattern-based methods [temperature gradient gel electrophoresis](https://bliptext.com/articles/temperature-gradient-gel-electrophoresis) (TGGE) and denaturing gradient gel electrophoresis (DGGE).\n# [TTGE profiles representing the bifidobacterial diversity of fecal samples from two healthy volunteers (A and B) before and after AMC (Oral Amoxicillin-Clavulanic Acid) treatment | upload.wikimedia.org/wikipedia/commons/2/2a////TTGE///profiles///representing///the///bifidobacterial///diversity///of///fecal///samples///journal///pone///0050257///g004.png]\n\n## Native\nNative gels are run in non-denaturing conditions so that the analyte's natural structure is maintained.\nThis allows the physical size of the folded or assembled complex to affect the mobility, allowing for analysis of all four levels of the biomolecular structure.\nFor biological samples, detergents are used only to the extent that they are necessary to [lyse](https://bliptext.com/articles/lysis) [lipid membranes](https://bliptext.com/articles/lipid-bilayer) in the [cell](https://bliptext.com/articles/cell-biology).\nComplexes remain—for the most part—associated and folded as they would be in the cell.\nOne downside, however, is that complexes may not separate cleanly or predictably, as it is difficult to predict how the molecule's shape and size will affect its mobility.\nAddressing and solving this problem is a major aim of [preparative native PAGE](https://bliptext.com/articles/qpnc-page).\nUnlike [denaturing](https://bliptext.com/articles/denaturation-biochemistry) methods, native gel electrophoresis does not use a charged [denaturing](https://bliptext.com/articles/denaturation-biochemistry) agent.\nThe molecules being separated (usually [proteins](https://bliptext.com/articles/proteins) or [nucleic acids](https://bliptext.com/articles/nucleic-acids)) therefore differ not only in [molecular mass](https://bliptext.com/articles/molecular-mass) and intrinsic charge, but also the cross-sectional area, and thus experience different electrophoretic forces dependent on the shape of the overall structure.\nFor proteins, since they remain in the native state they may be visualized not only by general protein staining reagents but also by specific enzyme-linked staining.\nA specific experiment example of an application of native gel electrophoresis is to check for enzymatic activity to verify the presence of the enzyme in the sample during protein purification.\nFor example, for the protein alkaline phosphatase, the staining solution is a mixture of 4-chloro-2-2methylbenzenediazonium salt with 3-phospho-2-naphthoic acid-2'-4'-dimethyl aniline in Tris buffer.\nThis stain is commercially sold as a kit for staining gels.\nIf the protein is present, the mechanism of the reaction takes place in the following order: it starts with the de-phosphorylation of 3-phospho-2-naphthoic acid-2'-4'-dimethyl aniline by alkaline phosphatase (water is needed for the reaction).\nThe phosphate group is released and replaced by an alcohol group from water.\nThe electrophile 4- chloro-2-2 methylbenzenediazonium (Fast Red TR Diazonium salt) displaces the alcohol group forming the final product Red Azo dye.\nAs its name implies, this is the final visible-red product of the reaction.\nIn undergraduate academic experimentation of protein purification, the gel is usually run next to commercial purified samples to visualize the results and conclude whether or not purification was successful.\n[Native](https://bliptext.com/articles/native-state) gel electrophoresis is typically used in [proteomics](https://bliptext.com/articles/proteomics) and [metallomics](https://bliptext.com/articles/metallome).\nHowever, native PAGE is also used to scan genes (DNA) for unknown mutations as in [single-strand conformation polymorphism](https://bliptext.com/articles/single-strand-conformation-polymorphism).\n# [Specific enzyme-linked staining: Glucose-6-Phosphate Dehydrogenase isoenzymes in Plasmodium falciparum infected Red blood cells | upload.wikimedia.org/wikipedia/commons/d/d8/Glucose-6-Phosphate///Dehydrogenase///activity///stain.jpg]\n\n# Buffers\nBuffers in gel electrophoresis are used to provide ions that carry a current and to maintain the pH at a relatively constant value.\nThese buffers have plenty of ions in them, which is necessary for the passage of electricity through them.\nSomething like distilled water or benzene contains few ions, which is not ideal for the use in electrophoresis.\nThere are a number of buffers used for electrophoresis.\nThe most common being, for nucleic acids [Tris/Acetate/EDTA](https://bliptext.com/articles/tae-buffer) (TAE), [Tris/Borate/EDTA](https://bliptext.com/articles/tbe-buffer) (TBE).\nMany other buffers have been proposed, e.g. [lithium borate](https://bliptext.com/articles/lb-buffer), which is rarely used, based on Pubmed citations (LB), isoelectric histidine, pK matched goods buffers, etc.; in most cases the purported rationale is lower current (less heat) matched ion mobilities, which leads to longer buffer life.\nBorate is problematic; Borate can polymerize, or interact with cis diols such as those found in RNA.\nTAE has the lowest buffering capacity but provides the best resolution for larger DNA.\nThis means a lower voltage and more time, but a better product.\nLB is relatively new and is ineffective in resolving fragments larger than 5 kbp; However, with its low conductivity, a much higher voltage could be used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis.\nAs low as one base pair size difference could be resolved in 3% agarose gel with an extremely low conductivity medium (1 mM Lithium borate).\nMost SDS-PAGE protein separations are performed using a [\"discontinuous\" (or DISC) buffer system](https://bliptext.com/articles/gel-electrophoresis-of-proteins) that significantly enhances the sharpness of the bands within the gel.\nDuring electrophoresis in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all of the proteins to focus on a single sharp band in a process called [isotachophoresis](https://bliptext.com/articles/isotachophoresis).\nSeparation of the proteins by size is achieved in the lower, \"resolving\" region of the gel.\nThe resolving gel typically has a much smaller pore size, which leads to a sieving effect that now determines the electrophoretic mobility of the proteins.\n\n# Visualization\nAfter the electrophoresis is complete, the molecules in the gel can be [stained](https://bliptext.com/articles/staining-biology) to make them visible.\nDNA may be visualized using [ethidium bromide](https://bliptext.com/articles/ethidium-bromide) which, when intercalated into DNA, [fluoresce](https://bliptext.com/articles/fluorescence) under [ultraviolet](https://bliptext.com/articles/ultraviolet) light, while protein may be visualised using [silver stain](https://bliptext.com/articles/silver-stain) or [Coomassie brilliant blue](https://bliptext.com/articles/coomassie-brilliant-blue) dye.\nOther methods may also be used to visualize the separation of the mixture's components on the gel.\nIf the molecules to be separated contain [radioactivity](https://bliptext.com/articles/radioactivity), for example in a [DNA sequencing](https://bliptext.com/articles/dna-sequencing) gel, an [autoradiogram](https://bliptext.com/articles/isotopic-tracer) can be recorded of the gel.\n[Photographs](https://bliptext.com/articles/photograph) can be taken of gels, often using a [Gel Doc](https://bliptext.com/articles/gel-doc) system.\nGels are then commonly labelled for presentation and scientific records on the popular figure-creation website, [SciUGo](https://bliptext.com/articles/undefined).\n\n# Downstream processing\nAfter separation, an additional separation method may then be used, such as [isoelectric focusing](https://bliptext.com/articles/isoelectric-focusing) or [SDS-PAGE](https://bliptext.com/articles/sds-page).\nThe gel will then be physically cut, and the protein complexes extracted from each portion separately.\nEach extract may then be analysed, such as by [peptide mass fingerprinting](https://bliptext.com/articles/peptide-mass-fingerprinting) or [de novo peptide sequencing](https://bliptext.com/articles/de-novo-peptide-sequencing) after [in-gel digestion](https://bliptext.com/articles/in-gel-digestion).\nThis can provide a great deal of information about the identities of the proteins in a complex.\n\n# Applications\nGel electrophoresis is used in [forensics](https://bliptext.com/articles/forensic-chemistry), [molecular biology](https://bliptext.com/articles/molecular-biology), [genetics](https://bliptext.com/articles/genetics), [microbiology](https://bliptext.com/articles/microbiology) and [biochemistry](https://bliptext.com/articles/biochemistry).\nThe results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device.\nThe 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.\nThe measurement and analysis are mostly done with specialized software.\nDepending 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.\n\n## Nucleic acids\nIn 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](https://bliptext.com/articles/sugar)-[phosphate](https://bliptext.com/articles/phosphate) backbone.\nDouble-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their size or, for cyclic fragments, their [radius of gyration](https://bliptext.com/articles/radius-of-gyration).\nCircular DNA such as [plasmids](https://bliptext.com/articles/plasmid), however, may show multiple bands, the speed of migration may depend on whether it is relaxed or supercoiled.\nSingle-stranded DNA or RNA tends to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure.\nTherefore, agents that disrupt the [hydrogen bonds](https://bliptext.com/articles/hydrogen-bond), such as [sodium hydroxide](https://bliptext.com/articles/sodium-hydroxide) or [formamide](https://bliptext.com/articles/formamide), are used to denature the nucleic acids and cause them to behave as long rods again.\nGel electrophoresis of large [DNA](https://bliptext.com/articles/dna) or [RNA](https://bliptext.com/articles/rna) is usually done by agarose gel electrophoresis.\nSee the \"[chain termination method](https://bliptext.com/articles/chain-termination-method)\" page for an example of a polyacrylamide DNA sequencing gel.\nCharacterization through ligand interaction of nucleic acids or fragments may be performed by mobility shift [affinity electrophoresis](https://bliptext.com/articles/affinity-electrophoresis).\nElectrophoresis of RNA samples can be used to check for genomic DNA contamination and also for RNA degradation.\nRNA from eukaryotic organisms shows distinct bands of 28s and 18s rRNA, the 28s band being approximately twice as intense as the 18s band.\nDegraded RNA has less sharply defined bands, has a smeared appearance, and the intensity ratio is less than 2:1.\n# [An agarose gel of a PCR product compared to a DNA ladder. | upload.wikimedia.org/wikipedia/commons/0/05////Pcr///gel.png]\n\n## Proteins\n[Proteins](https://bliptext.com/articles/protein), unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the polyacrylamide gel at similar rates, or all when placing a negative to positive EMF on the sample.\nProteins, therefore, are usually [denatured](https://bliptext.com/articles/denaturation-biochemistry) in the presence of a [detergent](https://bliptext.com/articles/detergent) such as [sodium dodecyl sulfate](https://bliptext.com/articles/sodium-dodecyl-sulfate) (SDS) that coats the proteins with a negative charge.\nGenerally, 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.\nSince 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 their size and not their charge or shape.\n[Proteins](https://bliptext.com/articles/protein) are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ([SDS-PAGE](https://bliptext.com/articles/sds-page)), by [native gel electrophoresis](https://bliptext.com/articles/native-gel-electrophoresis), by preparative [native gel electrophoresis](https://bliptext.com/articles/native-gel-electrophoresis) ([QPNC-PAGE](https://bliptext.com/articles/qpnc-page)), or by [2-D electrophoresis](https://bliptext.com/articles/2-d-electrophoresis).\nCharacterization through ligand interaction may be performed by [electroblotting](https://bliptext.com/articles/electroblotting) or by [affinity electrophoresis](https://bliptext.com/articles/affinity-electrophoresis) in agarose or by [capillary electrophoresis](https://bliptext.com/articles/capillary-electrophoresis) as for estimation of [binding constants](https://bliptext.com/articles/binding-constant) and determination of structural features like [glycan](https://bliptext.com/articles/glycan) content through [lectin](https://bliptext.com/articles/lectin) binding.\n# [SDS-PAGE autoradiography – The indicated proteins are present in different concentrations in the two samples. | upload.wikimedia.org/wikipedia/commons/3/32/SDSPAGE.png]\n\n## Nanoparticles\nA novel application for gel electrophoresis is the separation or characterization of metal or metal oxide nanoparticles (e.g. Au, Ag, ZnO, SiO2) regarding the size, shape, or surface chemistry of the nanoparticles.\nThe scope is to obtain a more homogeneous sample (e.g. narrower particle size distribution), which then can be used in further products/processes (e.g. self-assembly processes).\nFor the separation of nanoparticles within a gel, the key parameter is the ratio of the particle size to the mesh size, whereby two migration mechanisms were identified: the unrestricted mechanism, where the particle size << mesh size, and the restricted mechanism, where particle size is similar to mesh size.\n\n# History\nA 1959 book on electrophoresis by Milan Bier cites references from the 1800s.\nHowever, Oliver Smithies made significant contributions.\nBier states: \"The method of Smithies ... is finding wide application because of its unique separatory power.\"\nTaken in context, Bier clearly implies that Smithies' method is an improvement.","current_revision":null,"created_by":"kACnuQmH5k9Nr5VNVEpVeg7XpvUmMctD","created_at":"2025-01-25T17:23:37.946Z","updated_at":"2025-01-25T17:23:37.946Z","search_vector":"'electrophoresi':2 'gel':1","search_content":" \n \n**Name:** Gel electrophoresis \n**Classification:** [Electrophoresis](https://bliptext.com/articles/electrophoresis) \n**Related:** [Capillary electrophoresis](https://bliptext.com/articles/capillary-electrophoresis) [SDS-PAGE](https://bliptext.com/articles/polyacrylamide-gel-electrophoresis) [Two-dimensional gel electrophoresis](https://bliptext.com/articles/two-dimensional-gel-electrophoresis) [Temperature gradient gel electrophoresis](https://bliptext.com/articles/temperature-gradient-gel-electrophoresis) \n \n\n# \n**Gel electrophoresis** is a method for separation and analysis of [biomacromolecules](https://bliptext.com/articles/biomolecule) ([DNA](https://bliptext.com/articles/dna), [RNA](https://bliptext.com/articles/rna), [proteins](https://bliptext.com/articles/protein), etc.) and their fragments, based on their size and charge.\nIt is used in [clinical chemistry](https://bliptext.com/articles/clinical-chemistry) to separate proteins by charge or size (IEF agarose, essentially size independent) and in [biochemistry](https://bliptext.com/articles/biochemistry) and [molecular biology](https://bliptext.com/articles/molecular-biology) to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.\nNucleic acid molecules are separated by applying an [electric field](https://bliptext.com/articles/electric-field) to move the negatively charged molecules through a matrix of [agarose](https://bliptext.com/articles/agarose) or other substances.\nShorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel.\nThis phenomenon is called sieving.\nProteins are separated by the charge in agarose because the pores of the gel are too large to sieve proteins.\nGel electrophoresis can also be used for the separation of [nanoparticles](https://bliptext.com/articles/nanoparticle).\nGel electrophoresis uses a gel as an anticonvective medium or sieving medium during electrophoresis, the movement of a charged particle in an electric current.\nGels suppress the thermal convection caused by the application of the electric field, and can also act as a sieving medium, slowing the passage of molecules; gels can also simply serve to maintain the finished separation so that a post electrophoresis stain can be applied.\nDNA gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via [polymerase chain reaction](https://bliptext.com/articles/polymerase-chain-reaction) (PCR), but may be used as a preparative technique prior to use of other methods such as [mass spectrometry](https://bliptext.com/articles/mass-spectrometry), [RFLP](https://bliptext.com/articles/restriction-fragment-length-polymorphism), PCR, [cloning](https://bliptext.com/articles/cloning), [DNA sequencing](https://bliptext.com/articles/dna-sequencing), or [Southern blotting](https://bliptext.com/articles/southern-blot) for further characterization.\n# [The image above shows how small DNA fragments will migrate through agarose quickly but large size DNA fragments move more slowly during electrophoresis. The graph to the right shows the nonlinear relationship between the size of the DNA fragment and the distance migrated. | upload.wikimedia.org/wikipedia/commons/7/75////Gel///Electrophoresis.svg]\n# [Gel electrophoresis is a process where an electric current is applied to DNA samples creating fragments that can be used for comparison between DNA samples.\n \n DNA is extracted. \n Isolation and amplification of DNA. \n DNA added to the gel wells. \n Electric current applied to the gel. \n DNA bands are separated by size. \n DNA bands are stained. \n | upload.wikimedia.org/wikipedia/commons/d/d0/Gel///Electrophoresis///in///DNA///Fingerprinting.svg]\n\n# Physical basis\n[Electrophoresis](https://bliptext.com/articles/electrophoresis) is a process that enables the sorting of molecules based on charge, size, or shape.\nUsing an electric field, molecules (such as DNA) can be made to move through a gel made of [agarose](https://bliptext.com/articles/agarose) or [polyacrylamide](https://bliptext.com/articles/polyacrylamide).\nThe electric field consists of a negative charge at one end which pushes the molecules through the gel, and a positive charge at the other end that pulls the molecules through the gel.\nThe molecules being sorted are dispensed into a well in the gel material.\nThe gel is placed in an electrophoresis chamber, which is then connected to a power source.\nWhen the electric field is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster.\nThe different sized molecules form distinct bands on the gel.\nThe term \"[gel](https://bliptext.com/articles/gel)\" in this instance refers to the matrix used to contain, then separate the target molecules.\nIn most cases, the gel is a [crosslinked polymer](https://bliptext.com/articles/crosslinked-polymer) whose composition and porosity are chosen based on the specific weight and composition of the target to be analyzed.\nWhen separating [proteins](https://bliptext.com/articles/protein) or small [nucleic acids](https://bliptext.com/articles/nucleic-acid) ([DNA](https://bliptext.com/articles/dna), [RNA](https://bliptext.com/articles/rna), or [oligonucleotides](https://bliptext.com/articles/oligonucleotide)) the gel is usually composed of different concentrations of [acrylamide](https://bliptext.com/articles/acrylamide) and a [cross-linker](https://bliptext.com/articles/cross-linker), producing different sized mesh networks of poly[acrylamide](https://bliptext.com/articles/acrylamide).\nWhen separating larger nucleic acids (greater than a few hundred [bases](https://bliptext.com/articles/base-chemistry)), the preferred matrix is purified agarose.\nIn both cases, the gel forms a solid, yet porous matrix.\nAcrylamide, in contrast to polyacrylamide, is a [neurotoxin](https://bliptext.com/articles/neurotoxin) and must be handled using appropriate safety precautions to avoid poisoning.\nAgarose is composed of long unbranched chains of uncharged carbohydrates without cross-links resulting in a gel with large pores allowing for the separation of macromolecules and [macromolecular complexes](https://bliptext.com/articles/affinity-electrophoresis).\nElectrophoresis refers to the [electromotive force](https://bliptext.com/articles/electromotive-force) (EMF) that is used to move the molecules through the gel matrix.\nBy placing the molecules in wells in the gel and applying an electric field, the molecules will move through the matrix at different rates, determined largely by their mass when the charge-to-mass ratio (Z) of all species is uniform.\nHowever, when charges are not all uniform the electrical field generated by the electrophoresis procedure will cause the molecules to migrate differentially according to charge.\nSpecies that are net positively charged will migrate towards the [cathode](https://bliptext.com/articles/cathode) which is negatively charged (because this is an [electrolytic](https://bliptext.com/articles/electrolytic-cell) rather than [galvanic cell](https://bliptext.com/articles/galvanic-cell)), whereas species that are net negatively charged will migrate towards the positively charged anode.\nMass remains a factor in the speed with which these non-uniformly charged molecules migrate through the matrix toward their respective electrodes.\nIf several samples have been loaded into adjacent wells in the gel, they will run parallel in individual lanes.\nDepending on the number of different molecules, each lane shows the separation of the components from the original mixture as one or more distinct bands, one band per component.\nIncomplete separation of the components can lead to overlapping bands, or indistinguishable smears representing multiple unresolved components.\nBands in different lanes that end up at the same distance from the top contain molecules that passed through the gel at the same speed, which usually means they are approximately the same size.\nThere are [molecular weight size markers](https://bliptext.com/articles/molecular-weight-size-marker) available that contain a mixture of molecules of known sizes.\nIf 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 to determine their size.\nThe distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule (alternatively, this can be stated as the distance traveled is inversely proportional to the log of samples's molecular weight).\nThere are limits to electrophoretic techniques.\nSince passing a current through a gel causes heating, gels may melt during electrophoresis.\nElectrophoresis is performed in buffer solutions to reduce pH changes due to the electric field, which is important because the charge of DNA and RNA depends on pH, but running for too long can exhaust the buffering capacity of the solution.\nThere are also limitations in determining the molecular weight by SDS-PAGE, especially when trying to find the MW of an unknown protein.\nCertain biological variables are difficult or impossible to minimize and can affect electrophoretic migration.\nSuch factors include protein structure, post-translational modifications, and amino acid composition.\nFor example, tropomyosin is an acidic protein that migrates abnormally on SDS-PAGE gels.\nThis is because the acidic residues are repelled by the negatively charged SDS, leading to an inaccurate mass-to-charge ratio and migration.\nFurther, different preparations of genetic material may not migrate consistently with each other, for morphological or other reasons.\n# [Overview of gel electrophoresis. | upload.wikimedia.org/wikipedia/commons/4/46/SDS-PAGE///Electrophoresis.png]\n\n# Types of gel\nThe types of gel most typically used are agarose and polyacrylamide gels.\nEach type of gel is well-suited to different types and sizes of the analyte.\nPolyacrylamide gels are usually used for proteins and have very high resolving power for small fragments of DNA (5-500 bp).\nAgarose gels, on the other hand, have lower resolving power for DNA but have a greater range of separation, and are therefore used for DNA fragments of usually 50–20,000 bp in size, but the resolution of over 6 Mb is possible with [pulsed field gel electrophoresis](https://bliptext.com/articles/pulsed-field-gel-electrophoresis) (PFGE).\nPolyacrylamide gels are run in a vertical configuration while agarose gels are typically run horizontally in a submarine mode.\nThey also differ in their casting methodology, as agarose sets thermally, while polyacrylamide forms in a chemical polymerization reaction.\n\n## Agarose\nAgarose gels are made from the natural [polysaccharide](https://bliptext.com/articles/polysaccharide) [polymers](https://bliptext.com/articles/polymer) extracted from [seaweed](https://bliptext.com/articles/seaweed).\nAgarose gels are easily cast and handled compared to other matrices because the gel setting is a physical rather than chemical change.\nSamples are also easily recovered.\nAfter the experiment is finished, the resulting gel can be stored in a plastic bag in a refrigerator.\nAgarose gels do not have a uniform pore size, but are optimal for electrophoresis of proteins that are larger than 200 kDa.\nAgarose gel electrophoresis can also be used for the separation of DNA fragments ranging from 50 [base pair](https://bliptext.com/articles/base-pair) to several megabases (millions of bases), the largest of which require specialized apparatus.\nThe distance between DNA bands of different lengths is influenced by the percent agarose in the gel, with higher percentages requiring longer run times, sometimes days.\nInstead high percentage agarose gels should be run with a [pulsed field electrophoresis](https://bliptext.com/articles/pulsed-field-gel-electrophoresis) (PFE), or [field inversion electrophoresis](https://bliptext.com/articles/field-inversion-electrophoresis).\n\"Most agarose gels are made with between 0.7% (good separation or resolution of large 5–10kb DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose dissolved in electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1% gels are common for many applications.\"\n# [Inserting the gel comb in an agarose gel electrophoresis chamber | upload.wikimedia.org/wikipedia/commons/5/5b/Gel///electrophoresis///insert///comb.jpg]\n\n## Polyacrylamide\nPolyacrylamide gel electrophoresis (PAGE) is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel.\nPore size is controlled by modulating the concentrations of acrylamide and bis-acrylamide powder used in creating a gel.\nCare must be used when creating this type of gel, as acrylamide is a potent neurotoxin in its liquid and powdered forms.\nTraditional [DNA sequencing](https://bliptext.com/articles/dna-sequencing) techniques such as [Maxam-Gilbert](https://bliptext.com/articles/maxam-gilbert-sequencing) or [Sanger](https://bliptext.com/articles/dideoxy-termination) methods used polyacrylamide gels to separate DNA fragments differing by a single base-pair in length so the sequence could be read.\nMost modern DNA separation methods now use agarose gels, except for particularly small DNA fragments.\nIt is currently most often used in the field of [immunology](https://bliptext.com/articles/immunology) and protein analysis, often used to separate different proteins or [isoforms](https://bliptext.com/articles/isoforms) of the same protein into separate bands.\nThese can be transferred onto a [nitrocellulose](https://bliptext.com/articles/nitrocellulose) or [PVDF](https://bliptext.com/articles/pvdf) membrane to be probed with antibodies and corresponding markers, such as in a [western blot](https://bliptext.com/articles/western-blot).\nTypically [resolving gels](https://bliptext.com/articles/resolving-gels) are made in 6%, 8%, 10%, 12% or 15%.\nStacking gel (5%) is poured on top of the resolving gel and a gel comb (which forms the wells and defines the lanes where proteins, sample buffer, and ladders will be placed) is inserted.\nThe percentage chosen depends on the size of the protein that one wishes to identify or probe in the sample.\nThe smaller the known weight, the higher the percentage that should be used.\nChanges in the buffer system of the gel can help to further resolve proteins of very small sizes.\n\n## Starch\nPartially [hydrolysed](https://bliptext.com/articles/hydrolysis) potato starch makes for another non-toxic medium for protein electrophoresis.\nThe gels are slightly more opaque than acrylamide or agarose.\nNon-denatured proteins can be separated according to charge and size.\nThey are visualised using Napthal Black or Amido Black staining.\nTypical starch gel concentrations are 5% to 10%.\n\n# Gel conditions\n\n## Denaturing\n[Denaturing](https://bliptext.com/articles/denaturation-biochemistry) gels are run under conditions that disrupt the natural structure of the analyte, causing it to unfold into a linear chain.\nThus, the mobility of each [macromolecule](https://bliptext.com/articles/macromolecule) depends only on its linear length and its mass-to-charge ratio.\nThus, the secondary, tertiary, and quaternary levels of [biomolecular structure](https://bliptext.com/articles/biomolecular-structure) are disrupted, leaving only the primary structure to be analyzed.\nNucleic acids are often denatured by including [urea](https://bliptext.com/articles/urea) in the buffer, while proteins are denatured using [sodium dodecyl sulfate](https://bliptext.com/articles/sodium-dodecyl-sulfate), usually as part of the [SDS-PAGE](https://bliptext.com/articles/polyacrylamide-gel-electrophoresis) process.\nFor full denaturation of proteins, it is also necessary to reduce the covalent [disulfide bonds](https://bliptext.com/articles/disulfide-bond) that stabilize their [tertiary](https://bliptext.com/articles/tertiary-structure) and [quaternary structure](https://bliptext.com/articles/quaternary-structure), a method called reducing PAGE.\nReducing conditions are usually maintained by the addition of [beta-mercaptoethanol](https://bliptext.com/articles/beta-mercaptoethanol) or [dithiothreitol](https://bliptext.com/articles/dithiothreitol).\nFor a general analysis of protein samples, reducing PAGE is the most common form of [protein electrophoresis](https://bliptext.com/articles/protein-electrophoresis).\nDenaturing conditions are necessary for proper estimation of molecular weight of RNA.\nRNA is able to form more intramolecular interactions than DNA which may result in change of its [electrophoretic mobility](https://bliptext.com/articles/electrophoretic-mobility).\n[Urea](https://bliptext.com/articles/urea), [DMSO](https://bliptext.com/articles/dimethyl-sulfoxide) and [glyoxal](https://bliptext.com/articles/glyoxal) are the most often used denaturing agents to disrupt RNA structure.\nOriginally, highly toxic [methylmercury](https://bliptext.com/articles/methylmercury) hydroxide was often used in denaturing RNA electrophoresis, but it may be method of choice for some samples.\nDenaturing gel electrophoresis is used in the DNA and RNA banding pattern-based methods [temperature gradient gel electrophoresis](https://bliptext.com/articles/temperature-gradient-gel-electrophoresis) (TGGE) and denaturing gradient gel electrophoresis (DGGE).\n# [TTGE profiles representing the bifidobacterial diversity of fecal samples from two healthy volunteers (A and B) before and after AMC (Oral Amoxicillin-Clavulanic Acid) treatment | upload.wikimedia.org/wikipedia/commons/2/2a////TTGE///profiles///representing///the///bifidobacterial///diversity///of///fecal///samples///journal///pone///0050257///g004.png]\n\n## Native\nNative gels are run in non-denaturing conditions so that the analyte's natural structure is maintained.\nThis allows the physical size of the folded or assembled complex to affect the mobility, allowing for analysis of all four levels of the biomolecular structure.\nFor biological samples, detergents are used only to the extent that they are necessary to [lyse](https://bliptext.com/articles/lysis) [lipid membranes](https://bliptext.com/articles/lipid-bilayer) in the [cell](https://bliptext.com/articles/cell-biology).\nComplexes remain—for the most part—associated and folded as they would be in the cell.\nOne downside, however, is that complexes may not separate cleanly or predictably, as it is difficult to predict how the molecule's shape and size will affect its mobility.\nAddressing and solving this problem is a major aim of [preparative native PAGE](https://bliptext.com/articles/qpnc-page).\nUnlike [denaturing](https://bliptext.com/articles/denaturation-biochemistry) methods, native gel electrophoresis does not use a charged [denaturing](https://bliptext.com/articles/denaturation-biochemistry) agent.\nThe molecules being separated (usually [proteins](https://bliptext.com/articles/proteins) or [nucleic acids](https://bliptext.com/articles/nucleic-acids)) therefore differ not only in [molecular mass](https://bliptext.com/articles/molecular-mass) and intrinsic charge, but also the cross-sectional area, and thus experience different electrophoretic forces dependent on the shape of the overall structure.\nFor proteins, since they remain in the native state they may be visualized not only by general protein staining reagents but also by specific enzyme-linked staining.\nA specific experiment example of an application of native gel electrophoresis is to check for enzymatic activity to verify the presence of the enzyme in the sample during protein purification.\nFor example, for the protein alkaline phosphatase, the staining solution is a mixture of 4-chloro-2-2methylbenzenediazonium salt with 3-phospho-2-naphthoic acid-2'-4'-dimethyl aniline in Tris buffer.\nThis stain is commercially sold as a kit for staining gels.\nIf the protein is present, the mechanism of the reaction takes place in the following order: it starts with the de-phosphorylation of 3-phospho-2-naphthoic acid-2'-4'-dimethyl aniline by alkaline phosphatase (water is needed for the reaction).\nThe phosphate group is released and replaced by an alcohol group from water.\nThe electrophile 4- chloro-2-2 methylbenzenediazonium (Fast Red TR Diazonium salt) displaces the alcohol group forming the final product Red Azo dye.\nAs its name implies, this is the final visible-red product of the reaction.\nIn undergraduate academic experimentation of protein purification, the gel is usually run next to commercial purified samples to visualize the results and conclude whether or not purification was successful.\n[Native](https://bliptext.com/articles/native-state) gel electrophoresis is typically used in [proteomics](https://bliptext.com/articles/proteomics) and [metallomics](https://bliptext.com/articles/metallome).\nHowever, native PAGE is also used to scan genes (DNA) for unknown mutations as in [single-strand conformation polymorphism](https://bliptext.com/articles/single-strand-conformation-polymorphism).\n# [Specific enzyme-linked staining: Glucose-6-Phosphate Dehydrogenase isoenzymes in Plasmodium falciparum infected Red blood cells | upload.wikimedia.org/wikipedia/commons/d/d8/Glucose-6-Phosphate///Dehydrogenase///activity///stain.jpg]\n\n# Buffers\nBuffers in gel electrophoresis are used to provide ions that carry a current and to maintain the pH at a relatively constant value.\nThese buffers have plenty of ions in them, which is necessary for the passage of electricity through them.\nSomething like distilled water or benzene contains few ions, which is not ideal for the use in electrophoresis.\nThere are a number of buffers used for electrophoresis.\nThe most common being, for nucleic acids [Tris/Acetate/EDTA](https://bliptext.com/articles/tae-buffer) (TAE), [Tris/Borate/EDTA](https://bliptext.com/articles/tbe-buffer) (TBE).\nMany other buffers have been proposed, e.g. [lithium borate](https://bliptext.com/articles/lb-buffer), which is rarely used, based on Pubmed citations (LB), isoelectric histidine, pK matched goods buffers, etc.; in most cases the purported rationale is lower current (less heat) matched ion mobilities, which leads to longer buffer life.\nBorate is problematic; Borate can polymerize, or interact with cis diols such as those found in RNA.\nTAE has the lowest buffering capacity but provides the best resolution for larger DNA.\nThis means a lower voltage and more time, but a better product.\nLB is relatively new and is ineffective in resolving fragments larger than 5 kbp; However, with its low conductivity, a much higher voltage could be used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis.\nAs low as one base pair size difference could be resolved in 3% agarose gel with an extremely low conductivity medium (1 mM Lithium borate).\nMost SDS-PAGE protein separations are performed using a [\"discontinuous\" (or DISC) buffer system](https://bliptext.com/articles/gel-electrophoresis-of-proteins) that significantly enhances the sharpness of the bands within the gel.\nDuring electrophoresis in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all of the proteins to focus on a single sharp band in a process called [isotachophoresis](https://bliptext.com/articles/isotachophoresis).\nSeparation of the proteins by size is achieved in the lower, \"resolving\" region of the gel.\nThe resolving gel typically has a much smaller pore size, which leads to a sieving effect that now determines the electrophoretic mobility of the proteins.\n\n# Visualization\nAfter the electrophoresis is complete, the molecules in the gel can be [stained](https://bliptext.com/articles/staining-biology) to make them visible.\nDNA may be visualized using [ethidium bromide](https://bliptext.com/articles/ethidium-bromide) which, when intercalated into DNA, [fluoresce](https://bliptext.com/articles/fluorescence) under [ultraviolet](https://bliptext.com/articles/ultraviolet) light, while protein may be visualised using [silver stain](https://bliptext.com/articles/silver-stain) or [Coomassie brilliant blue](https://bliptext.com/articles/coomassie-brilliant-blue) dye.\nOther methods may also be used to visualize the separation of the mixture's components on the gel.\nIf the molecules to be separated contain [radioactivity](https://bliptext.com/articles/radioactivity), for example in a [DNA sequencing](https://bliptext.com/articles/dna-sequencing) gel, an [autoradiogram](https://bliptext.com/articles/isotopic-tracer) can be recorded of the gel.\n[Photographs](https://bliptext.com/articles/photograph) can be taken of gels, often using a [Gel Doc](https://bliptext.com/articles/gel-doc) system.\nGels are then commonly labelled for presentation and scientific records on the popular figure-creation website, [SciUGo](https://bliptext.com/articles/undefined).\n\n# Downstream processing\nAfter separation, an additional separation method may then be used, such as [isoelectric focusing](https://bliptext.com/articles/isoelectric-focusing) or [SDS-PAGE](https://bliptext.com/articles/sds-page).\nThe gel will then be physically cut, and the protein complexes extracted from each portion separately.\nEach extract may then be analysed, such as by [peptide mass fingerprinting](https://bliptext.com/articles/peptide-mass-fingerprinting) or [de novo peptide sequencing](https://bliptext.com/articles/de-novo-peptide-sequencing) after [in-gel digestion](https://bliptext.com/articles/in-gel-digestion).\nThis can provide a great deal of information about the identities of the proteins in a complex.\n\n# Applications\nGel electrophoresis is used in [forensics](https://bliptext.com/articles/forensic-chemistry), [molecular biology](https://bliptext.com/articles/molecular-biology), [genetics](https://bliptext.com/articles/genetics), [microbiology](https://bliptext.com/articles/microbiology) and [biochemistry](https://bliptext.com/articles/biochemistry).\nThe results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device.\nThe 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.\nThe measurement and analysis are mostly done with specialized software.\nDepending 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.\n\n## Nucleic acids\nIn 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](https://bliptext.com/articles/sugar)-[phosphate](https://bliptext.com/articles/phosphate) backbone.\nDouble-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their size or, for cyclic fragments, their [radius of gyration](https://bliptext.com/articles/radius-of-gyration).\nCircular DNA such as [plasmids](https://bliptext.com/articles/plasmid), however, may show multiple bands, the speed of migration may depend on whether it is relaxed or supercoiled.\nSingle-stranded DNA or RNA tends to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure.\nTherefore, agents that disrupt the [hydrogen bonds](https://bliptext.com/articles/hydrogen-bond), such as [sodium hydroxide](https://bliptext.com/articles/sodium-hydroxide) or [formamide](https://bliptext.com/articles/formamide), are used to denature the nucleic acids and cause them to behave as long rods again.\nGel electrophoresis of large [DNA](https://bliptext.com/articles/dna) or [RNA](https://bliptext.com/articles/rna) is usually done by agarose gel electrophoresis.\nSee the \"[chain termination method](https://bliptext.com/articles/chain-termination-method)\" page for an example of a polyacrylamide DNA sequencing gel.\nCharacterization through ligand interaction of nucleic acids or fragments may be performed by mobility shift [affinity electrophoresis](https://bliptext.com/articles/affinity-electrophoresis).\nElectrophoresis of RNA samples can be used to check for genomic DNA contamination and also for RNA degradation.\nRNA from eukaryotic organisms shows distinct bands of 28s and 18s rRNA, the 28s band being approximately twice as intense as the 18s band.\nDegraded RNA has less sharply defined bands, has a smeared appearance, and the intensity ratio is less than 2:1.\n# [An agarose gel of a PCR product compared to a DNA ladder. | upload.wikimedia.org/wikipedia/commons/0/05////Pcr///gel.png]\n\n## Proteins\n[Proteins](https://bliptext.com/articles/protein), unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the polyacrylamide gel at similar rates, or all when placing a negative to positive EMF on the sample.\nProteins, therefore, are usually [denatured](https://bliptext.com/articles/denaturation-biochemistry) in the presence of a [detergent](https://bliptext.com/articles/detergent) such as [sodium dodecyl sulfate](https://bliptext.com/articles/sodium-dodecyl-sulfate) (SDS) that coats the proteins with a negative charge.\nGenerally, 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.\nSince 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 their size and not their charge or shape.\n[Proteins](https://bliptext.com/articles/protein) are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ([SDS-PAGE](https://bliptext.com/articles/sds-page)), by [native gel electrophoresis](https://bliptext.com/articles/native-gel-electrophoresis), by preparative [native gel electrophoresis](https://bliptext.com/articles/native-gel-electrophoresis) ([QPNC-PAGE](https://bliptext.com/articles/qpnc-page)), or by [2-D electrophoresis](https://bliptext.com/articles/2-d-electrophoresis).\nCharacterization through ligand interaction may be performed by [electroblotting](https://bliptext.com/articles/electroblotting) or by [affinity electrophoresis](https://bliptext.com/articles/affinity-electrophoresis) in agarose or by [capillary electrophoresis](https://bliptext.com/articles/capillary-electrophoresis) as for estimation of [binding constants](https://bliptext.com/articles/binding-constant) and determination of structural features like [glycan](https://bliptext.com/articles/glycan) content through [lectin](https://bliptext.com/articles/lectin) binding.\n# [SDS-PAGE autoradiography – The indicated proteins are present in different concentrations in the two samples. | upload.wikimedia.org/wikipedia/commons/3/32/SDSPAGE.png]\n\n## Nanoparticles\nA novel application for gel electrophoresis is the separation or characterization of metal or metal oxide nanoparticles (e.g. Au, Ag, ZnO, SiO2) regarding the size, shape, or surface chemistry of the nanoparticles.\nThe scope is to obtain a more homogeneous sample (e.g. narrower particle size distribution), which then can be used in further products/processes (e.g. self-assembly processes).\nFor the separation of nanoparticles within a gel, the key parameter is the ratio of the particle size to the mesh size, whereby two migration mechanisms were identified: the unrestricted mechanism, where the particle size << mesh size, and the restricted mechanism, where particle size is similar to mesh size.\n\n# History\nA 1959 book on electrophoresis by Milan Bier cites references from the 1800s.\nHowever, Oliver Smithies made significant contributions.\nBier states: \"The method of Smithies ... is finding wide application because of its unique separatory power.\"\nTaken in context, Bier clearly implies that Smithies' method is an improvement.","revisionCount":0},"hasMore":false,"nextCursor":null}