What is the relationship between the size of a DNA fragment and the distance it migrates in the jail?

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Method for separation and analysis of biomolecules

Gel electrophoresis

Gel electrophoresis apparatus – an agarose gel is placed in this buffer-filled box and an 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 positively charged anode (red wire).

ClassificationElectrophoresisOther techniquesRelatedCapillary electrophoresis
SDS-PAGE
Two-dimensional gel electrophoresis
Temperature gradient gel electrophoresis

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.

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. # 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.

Gel electrophoresis is a method for separation and analysis of biomacromolecules (DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF agarose, essentially size independent) and in biochemistry and 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.[1]

Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through a matrix of agarose or other substances. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving.[2] Proteins are separated by the charge in agarose because the pores of the gel are too small to sieve proteins. Gel electrophoresis can also be used for the separation of nanoparticles.

Gel electrophoresis uses a gel as an anticonvective medium or sieving medium during electrophoresis, the movement of a charged particle in an electrical current. Gels 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.[3] DNA gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via polymerase chain reaction (PCR), 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.

Physical basis

See also: electrophoresis

Overview of gel electrophoresis.

Electrophoresis is a process that enables the sorting of molecules based on size. Using an electric field, molecules (such as DNA) can be made to move through a gel made of agarose or polyacrylamide. The 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. The molecules being sorted are dispensed into a well in the gel material. The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric field is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel.[4]

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 are 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 appropriate safety precautions to avoid poisoning. Agarose 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.[5]

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 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. However, when charges are not all uniform the electrical field generated by the electrophoresis procedure will cause the molecules to migrate differentially according to charge. Species that are net positively charged will migrate towards the cathode which is negatively charged (because this is an electrolytic rather than galvanic cell), whereas species that are net negatively charged will migrate towards the positively charged anode. Mass remains a factor in the speed with which these non-uniformly charged molecules migrate through the matrix toward their respective electrodes.[6]

If several samples have been loaded into adjacent wells in the gel, they will run parallel in individual lanes. Depending 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. Incomplete separation of the components can lead to overlapping bands, or indistinguishable smears representing multiple unresolved components.[citation needed] Bands 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. 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 to determine their size. The 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).[7]

There are limits to electrophoretic techniques. Since passing a current through a gel causes heating, gels may melt during electrophoresis. Electrophoresis 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. There are also limitations in determining the molecular weight by SDS-PAGE, especially when trying to find the MW of an unknown protein. Certain biological variables are difficult or impossible to minimize and can affect electrophoretic migration. Such factors include protein structure, post-translational modifications, and amino acid composition. For example, tropomyosin is an acidic protein that migrates abnormally on SDS-PAGE gels. This is because the acidic residues are repelled by the negatively charged SDS, leading to an inaccurate mass-to-charge ratio and migration.[8] Further, different preparations of genetic material may not migrate consistently with each other, for morphological or other reasons.

Types of gel

The types of gel most typically used are agarose and polyacrylamide gels. Each type of gel is well-suited to different types and sizes of the analyte. Polyacrylamide gels are usually used for proteins and have very high resolving power for small fragments of DNA (5-500 bp). Agarose 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 (PFGE).[9] Polyacrylamide gels are run in a vertical configuration while agarose gels are typically run horizontally in a submarine mode. They also differ in their casting methodology, as agarose sets thermally, while polyacrylamide forms in a chemical polymerization reaction.

Agarose

Inserting the gel comb in an agarose gel electrophoresis chamber

Main article: Agarose gel electrophoresis

Agarose gels are made from the natural polysaccharide polymers extracted from seaweed. Agarose gels are easily cast and handled compared to other matrices because the gel setting is a physical rather than chemical change. Samples are also easily recovered. After the experiment is finished, the resulting gel can be stored in a plastic bag in a refrigerator.

Agarose gels do not have a uniform pore size, but are optimal for electrophoresis of proteins that are larger than 200 kDa.[10] Agarose gel electrophoresis can also be used for the separation of DNA fragments ranging from 50 base pair to several megabases (millions of bases)[citation needed], the largest of which require specialized apparatus. The 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. Instead high percentage agarose gels should be run with a pulsed field electrophoresis (PFE), or field inversion electrophoresis.

"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."[11]

Polyacrylamide

Main article: Polyacrylamide gel electrophoresis

Polyacrylamide 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. Pore size is controlled by modulating the concentrations of acrylamide and bis-acrylamide powder used in creating a gel. Care must be used when creating this type of gel, as acrylamide is a potent neurotoxin in its liquid and powdered forms.

Traditional DNA sequencing techniques such as Maxam-Gilbert or Sanger methods used polyacrylamide gels to separate DNA fragments differing by a single base-pair in length so the sequence could be read. Most modern DNA separation methods now use agarose gels, except for particularly small DNA fragments. It is currently most often used in the field of immunology and protein analysis, often used to separate different proteins or isoforms of the same protein into separate bands. These can be transferred onto a nitrocellulose or PVDF membrane to be probed with antibodies and corresponding markers, such as in a western blot.

Typically resolving gels are made in 6%, 8%, 10%, 12% or 15%. Stacking 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. The percentage chosen depends on the size of the protein that one wishes to identify or probe in the sample. The smaller the known weight, the higher the percentage that should be used. Changes in the buffer system of the gel can help to further resolve proteins of very small sizes.[12]

Starch

Partially hydrolysed potato starch makes for another non-toxic medium for protein electrophoresis. The gels are slightly more opaque than acrylamide or agarose. Non-denatured proteins can be separated according to charge and size. They are visualised using Napthal Black or Amido Black staining. Typical starch gel concentrations are 5% to 10%.[13][14][15]

Gel conditions

Denaturing

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

Denaturing gels are run under conditions that disrupt the natural structure of the analyte, causing it to unfold into a linear chain. Thus, the mobility of each macromolecule depends only on its linear length and its mass-to-charge ratio. Thus, the secondary, tertiary, and quaternary levels of biomolecular structure are disrupted, leaving only the primary structure to be analyzed.

Nucleic acids are often denatured by including urea in the buffer, while proteins are denatured using sodium dodecyl sulfate, usually as part of the SDS-PAGE process. For full denaturation of proteins, it is also necessary to reduce the covalent disulfide bonds that stabilize their tertiary and quaternary structure, a method called reducing PAGE. Reducing conditions are usually maintained by the addition of beta-mercaptoethanol or dithiothreitol. For a general analysis of protein samples, reducing PAGE is the most common form of protein electrophoresis.

Denaturing conditions are necessary for proper estimation of molecular weight of RNA. RNA is able to form more intramolecular interactions than DNA which may result in change of its electrophoretic mobility. Urea, DMSO and glyoxal are the most often used denaturing agents to disrupt RNA structure. Originally, highly toxic methylmercury hydroxide was often used in denaturing RNA electrophoresis,[16] but it may be method of choice for some samples.[17]

Denaturing gel electrophoresis is used in the DNA and RNA banding pattern-based methods temperature gradient gel electrophoresis (TGGE)[18] and denaturing gradient gel electrophoresis (DGGE).[19]

Native

Specific enzyme-linked staining: Glucose-6-Phosphate Dehydrogenase isoenzymes in Plasmodium falciparum infected Red blood cells[20]

Native gels are run in non-denaturing conditions so that the analyte's natural structure is maintained. This 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. For biological samples, detergents are used only to the extent that they are necessary to lyse lipid membranes in the cell. Complexes remain—for the most part—associated and folded as they would be in the cell. One 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. Addressing and solving this problem is a major aim of quantitative native PAGE.

Unlike denaturing methods, native gel electrophoresis does not use a charged denaturing agent. The molecules being separated (usually proteins or nucleic acids) therefore differ not only in 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. For 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.

A 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. For 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. This stain is commercially sold as a kit for staining gels. If 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). The phosphate group is released and replaced by an alcohol group from water. The electrophile 4- chloro-2-2 methylbenzenediazonium (Fast Red TR Diazonium salt) displaces the alcohol group forming the final product Red Azo dye. As its name implies, this is the final visible-red product of the reaction. In 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.[21]

Native gel electrophoresis is typically used in proteomics and metallomics. However, native PAGE is also used to scan genes (DNA) for unknown mutations as in Single-strand conformation polymorphism.

Buffers

Buffers in gel electrophoresis are used to provide ions that carry a current and to maintain the pH at a relatively constant value. These buffers have plenty of ions in them, which is necessary for the passage of electricity through them. Something like distilled water or benzene contains few ions, which is not ideal for the use in electrophoresis.[22] There are a number of buffers used for electrophoresis. The most common being, for nucleic acids Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA (TBE). Many other buffers have been proposed, e.g. lithium borate, 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. Borate is problematic; Borate can polymerize, or interact with cis diols such as those found in RNA. TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product. LB 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. As low as one base pair size difference could be resolved in 3% agarose gel with an extremely low conductivity medium (1 mM Lithium borate).[23]

Most SDS-PAGE protein separations are performed using a "discontinuous" (or DISC) buffer system that significantly enhances the sharpness of the bands within the gel. During 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. Separation of the proteins by size is achieved in the lower, "resolving" region of the gel. The resolving gel typically has a much smaller pore size, which leads to a sieving effect that now determines the electrophoretic mobility of the proteins.

Visualization

Further information: Gel electrophoresis of nucleic acids § Visualization, and Gel electrophoresis of proteins § Visualization

After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. DNA may be visualized using ethidium bromide which, when intercalated into DNA, fluoresce under ultraviolet light, while protein may be visualised using silver stain or Coomassie brilliant blue dye. Other methods may also be used to visualize the separation of the mixture's components on the gel. If the molecules to be separated contain radioactivity, for example in a DNA sequencing gel, an autoradiogram can be recorded of the gel. Photographs can be taken of gels, often using a Gel Doc system.

Downstream processing

After separation, an additional separation method may then be used, such as isoelectric focusing or SDS-PAGE. The gel will then be physically cut, and the protein complexes extracted from each portion separately. Each extract may then be analysed, such as by peptide mass fingerprinting or de novo peptide sequencing after in-gel digestion. This can provide a great deal of information about the identities of the proteins in a complex.

Applications

• Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction mapping of cloned DNA.
• Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
• Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer.

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.

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

Main article: Gel electrophoresis of nucleic acids

An agarose gel of a PCR product compared to a DNA ladder.

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.[24]

Double-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. Circular DNA such as plasmids, however, may show multiple bands, the speed of migration may depend on whether it is relaxed or supercoiled. Single-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. 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.[25]

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. Characterization through ligand interaction of nucleic acids or fragments may be performed by mobility shift affinity electrophoresis.

Electrophoresis of RNA samples can be used to check for genomic DNA contamination and also for RNA degradation. RNA from eukaryotic organisms shows distinct bands of 28s and 18s rRNA, the 28s band being approximately twice as intense as the 18s band. Degraded RNA has less sharply defined bands, has a smeared appearance, and the intensity ratio is less than 2:1.

Proteins

Main article: Gel electrophoresis of proteins

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 polyacrylamide gel at similar rates, or 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 (SDS) that coats the proteins with a negative charge.[3] 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 their size and not their charge or shape.[3]

Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), by native gel electrophoresis, by preparative gel electrophoresis (QPNC-PAGE), or by 2-D electrophoresis.

Characterization through ligand interaction may be performed by electroblotting or by affinity electrophoresis in agarose or by capillary electrophoresis as for estimation of binding constants and determination of structural features like glycan content through lectin binding.

Nanoparticles

A novel application for gel electrophoresis is to separate or characterize metal or metal oxide nanoparticles (e.g. Au, Ag, ZnO, SiO2) regarding the size, shape, or surface chemistry of the nanoparticles. The 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). For the separation of nanoparticles within a gel, the particle size about the mesh size is the key parameter, 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.[26]

History

• 1930s – first reports of the use of sucrose for gel electrophoresis
• 1955 – introduction of starch gels, mediocre separation (Smithies)[14]
• 1959 – introduction of acrylamide gels; disc electrophoresis (Ornstein and Davis); accurate control of parameters such as pore size and stability; and (Raymond and Weintraub)
• 1966 – first use of agar gels[27]
• 1969 – introduction of denaturing agents especially SDS separation of protein subunit (Weber and Osborn)[28]
• 1970 – Laemmli separated 28 components of T4 phage using a stacking gel and SDS
• 1972 – agarose gels with ethidium bromide stain[29]
• 1975 – 2-dimensional gels (O’Farrell); isoelectric focusing then SDS gel electrophoresis
• 1977 – sequencing gels
• 1983 – pulsed field gel electrophoresis enables separation of large DNA molecules
• 1983 – introduction of capillary electrophoresis
• 2004 – introduction of a standardized time of polymerization of acrylamide gels enables clean and predictable separation of native proteins (Kastenholz)[30]

A 1959 book on electrophoresis by Milan Bier cites references from the 1800s.[31] 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.

See also

• History of electrophoresis
• Electrophoretic mobility shift assay
• Gel extraction
• Isoelectric focusing
• Pulsed field gel electrophoresis
• Nonlinear frictiophoresis
• Two-dimensional gel electrophoresis
• SDD-AGE
• Zymography
• Fast parallel proteolysis[32]

References

1. ^ Kryndushkin DS, Alexandrov IM, Ter-Avanesyan MD, Kushnirov VV (2003). "Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104". J Biol Chem. 278 (49): 49636–43. doi:10.1074/jbc.M307996200. PMID 14507919.{{cite journal}}: CS1 maint: multiple names: authors list (link)
2. ^ Sambrook, Joseph (2001). Molecular cloning : a laboratory manual (in Spanish). Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press. ISBN 978-0-87969-576-7. OCLC 45015638.
3. ^ a b c Berg, Jeremy (2002). Biochemistry (in Estonian). New York: W.H. Freeman. ISBN 978-0-7167-4955-4. OCLC 48055706.
4. ^ Wilson, Keith (2018). Wilson and Walker's principles and techniques of biochemistry and molecular biology. Cambridge, United Kingdom New York, NY: Cambridge University Press. ISBN 978-1-316-61476-1. OCLC 998750377.
5. ^ Boyer, Rodney (2000). Modern experimental biochemistry (in Estonian). San Francisco: Benjamin Cummings. ISBN 978-0-8053-3111-0. OCLC 44493241.
6. ^ Robyt, John (1990). Biochemical techniques : theory and practice. Prospect Heights, Ill: Waveland Press. ISBN 978-0-88133-556-9. OCLC 22549624.
7. ^ Lee PY, Costumbrado J, Hsu CY, Kim YH (2012). "Agarose gel electrophoresis for the separation of DNA fragments". J Vis Exp (62). doi:10.3791/3923. PMC 4846332. PMID 22546956.{{cite journal}}: CS1 maint: multiple names: authors list (link)
8. ^ "Molecular Weight Determination by SDS-PAGE, Rev B" (PDF). www.bio-rad.com. Retrieved 23 March 2022.
9. ^ Tom Maniatis; E. F. Fritsch; Joseph Sambrook (1982). "Chapter 5, protocol 1". Molecular Cloning - A Laboratory Manual. Vol. 1 (3rd ed.). p. 5.2–5.3. ISBN 978-0879691363.
10. ^ Smisek, David L.; Hoagland, David A. (1989). "Agarose gel electrophoresis of high molecular weight, synthetic polyelectrolytes". Macromolecules. American Chemical Society (ACS). 22 (5): 2270–2277. Bibcode:1989MaMol..22.2270S. doi:10.1021/ma00195a048. ISSN 0024-9297.
11. ^ "Agarose gel electrophoresis (basic method)". Biological Protocols. Retrieved 23 March 2022.
12. ^ Schägger H (2006). "Tricine-SDS-PAGE". Nat Protoc. 1 (1): 16–22. doi:10.1038/nprot.2006.4. PMID 17406207. S2CID 209529082.
13. ^ Gordon, A.H. (1969). Electrophoresis of Proteins in Polyacrylamide and Starch Gels: Laboratory Techniques in Biochemistry and Molecular Biology. Amsterdam: North-Holland Pub. Co. ISBN 978-0-7204-4202-1. OCLC 21766.
14. ^ a b Smithies O (1955). "Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults". Biochem J. 61 (4): 629–41. doi:10.1042/bj0610629. PMC 1215845. PMID 13276348.
15. ^ Wraxall BG, Culliford BJ (1968). "A thin-layer starch gel method for enzyme typing of bloodstains". J Forensic Sci Soc. 8 (2): 81–2. doi:10.1016/s0015-7368(68)70449-7. PMID 5738223.
16. ^ Buell GN, Wickens MP, Payvar F, Schimke RT (1978). "Synthesis of full length cDNAs from four partially purified oviduct mRNAs". J Biol Chem. 253 (7): 2471–82. doi:10.1016/S0021-9258(17)38097-3. PMID 632280.{{cite journal}}: CS1 maint: multiple names: authors list (link)
17. ^ Schelp C, Kaaden OR (1989). "Enhanced full-length transcription of Sindbis virus RNA by effective denaturation with methylmercury hydroxide". Acta Virol. 33 (3): 297–302. PMID 2570517.
18. ^ Fromin N, Hamelin J, Tarnawski S, Roesti D, Jourdain-Miserez K, Forestier N; et al. (2002). "Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns". Environ Microbiol. 4 (11): 634–43. doi:10.1046/j.1462-2920.2002.00358.x. PMID 12460271.{{cite journal}}: CS1 maint: multiple names: authors list (link)
19. ^ Fischer SG, Lerman LS (1979). "Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis". Cell. 16 (1): 191–200. doi:10.1016/0092-8674(79)90200-9. PMID 369706. S2CID 9369012.
20. ^ Hempelmann E, Wilson RJ (1981). "Detection of glucose-6-phosphate dehydrogenase in malarial parasites". Mol Biochem Parasitol. 2 (3–4): 197–204. doi:10.1016/0166-6851(81)90100-6. PMID 7012616.
21. ^ Ninfa AJ, Ballou DP (1998). Fundamental Approaches to Biochemistry and Biotechnology. Bethesda, Md: Fitzgerald Science Press. ISBN 9781891786006.
22. ^ Ninfa, Alexander J.; Ballou, David P.; Benore, Marilee (2009). fundamental laboratory approaches for biochemistry and biotechnology. Hoboken, NJ: Wiley. p. 161. ISBN 978-0470087664.
23. ^ Brody JR, Kern SE (2004). "History and principles of conductive media for standard DNA electrophoresis". Anal Biochem. 333 (1): 1–13. doi:10.1016/j.ab.2004.05.054. PMID 15351274.
24. ^ Lodish H; Berk A; Matsudaira P (2004). Molecular Cell Biology (5th ed.). WH Freeman: New York, NY. ISBN 978-0-7167-4366-8.
25. ^ Troubleshooting DNA agarose gel electrophoresis. Focus 19:3 p.66 (1997).
26. ^ Barasinski, Matthäus; Garnweitner, Georg (12 February 2020). "Restricted and Unrestricted Migration Mechanisms of Silica Nanoparticles in Agarose Gels and Their Utilization for the Separation of Binary Mixtures". The Journal of Physical Chemistry C. American Chemical Society (ACS). 124 (9): 5157–5166. doi:10.1021/acs.jpcc.9b10644. ISSN 1932-7447. S2CID 213566317.
27. ^ Thorne HV (1966). "Electrophoretic separation of polyoma virus DNA from host cell DNA". Virology. 29 (2): 234–9. doi:10.1016/0042-6822(66)90029-8. PMID 4287545.
28. ^ Weber K, Osborn M (1969). "The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis". J Biol Chem. 244 (16): 4406–12. doi:10.1016/S0021-9258(18)94333-4. PMID 5806584.
29. ^ Aaij C, Borst P (1972). "The gel electrophoresis of DNA". Biochim Biophys Acta. 269 (2): 192–200. doi:10.1016/0005-2787(72)90426-1. PMID 5063906.
30. ^ Kastenholz, Bernd (2004). "Preparative Native Continuous Polyacrylamide Gel Electrophoresis (PNC‐PAGE): An Efficient Method for Isolating Cadmium Cofactors in Biological Systems". Analytical Letters. Informa UK Limited. 37 (4): 657–665. doi:10.1081/al-120029742. ISSN 0003-2719. S2CID 97636537.
31. ^ Bier, Milan (1959). Electrophoresis: theory, methods, and applications. Academic Press. p. 225. OCLC 1175404.
32. ^ Minde, David P.; Maurice, Madelon M.; Rüdiger, Stefan G. D. (3 October 2012). Uversky, Vladimir N. (ed.). "Determining Biophysical Protein Stability in Lysates by a Fast Proteolysis Assay, FASTpp". PLOS ONE. Public Library of Science (PLoS). 7 (10): e46147. Bibcode:2012PLoSO...746147M. doi:10.1371/journal.pone.0046147. ISSN 1932-6203. PMC 3463568. PMID 23056252.

External links

Wikimedia Commons has media related to Gel electrophoresis.

• Biotechniques Laboratory electrophoresis demonstration, from the University of Utah's Genetic Science Learning Center
• Discontinuous native protein gel electrophoresis
• Drinking straw electrophoresis
• How to run a DNA or RNA gel
• Animation of gel analysis of DNA restriction
• Step by step photos of running a gel and extracting DNA
• A typical method from wikiversity

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Page 2

2D-Gels (Coomassie stained)

Robots are used for the isolation of protein spots from 2D gels in modern laboratories.

Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell[1] and Klose[2] in 1975.

Basis for separation

2-D electrophoresis begins with electrophoresis in the first dimension and then separates the molecules perpendicularly from the first to create an electropherogram in the second dimension. In electrophoresis in the first dimension, molecules are separated linearly according to their isoelectric point. In the second dimension, the molecules are then separated at 90 degrees from the first electropherogram according to molecular mass. Since it is unlikely that two molecules will be similar in two distinct properties, molecules are more effectively separated in 2-D electrophoresis than in 1-D electrophoresis.

The two dimensions that proteins are separated into using this technique can be isoelectric point, protein complex mass in the native state, or protein mass.

Separation of the proteins by isoelectric point is called isoelectric focusing (IEF). Thereby, a pH gradient is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pH values other than their isoelectric point, proteins will be charged. If they are positively charged, they will be pulled towards the more negative end of the gel and if they are negatively charged they will be pulled to the more positive end of the gel. The proteins applied in the first dimension will move along the gel and will accumulate at their isoelectric point; that is, the point at which the overall charge on the protein is 0 (a neutral charge).

For the analysis of the functioning of proteins in a cell, the knowledge of their cooperation is essential. Most often proteins act together in complexes to be fully functional. The analysis of this sub organelle organisation of the cell requires techniques conserving the native state of the protein complexes. In native polyacrylamide gel electrophoresis (native PAGE), proteins remain in their native state and are separated in the electric field following their mass and the mass of their complexes respectively. To obtain a separation by size and not by net charge, as in IEF, an additional charge is transferred to the proteins by the use of Coomassie brilliant blue or lithium dodecyl sulfate. After completion of the first dimension the complexes are destroyed by applying the denaturing SDS-PAGE in the second dimension, where the proteins of which the complexes are composed of are separated by their mass.

Before separating the proteins by mass, they are treated with sodium dodecyl sulfate (SDS) along with other reagents (SDS-PAGE in 1-D). This denatures the proteins (that is, it unfolds them into long, straight molecules) and binds a number of SDS molecules roughly proportional to the protein's length. Because a protein's length (when unfolded) is roughly proportional to its mass, this is equivalent to saying that it attaches a number of SDS molecules roughly proportional to the protein's mass. Since the SDS molecules are negatively charged, the result of this is that all of the proteins will have approximately the same mass-to-charge ratio as each other. In addition, proteins will not migrate when they have no charge (a result of the isoelectric focusing step) therefore the coating of the protein in SDS (negatively charged) allows migration of the proteins in the second dimension (SDS-PAGE, it is not compatible for use in the first dimension as it is charged and a nonionic or zwitterionic detergent needs to be used). In the second dimension, an electric potential is again applied, but at a 90 degree angle from the first field. The proteins will be attracted to the more positive side of the gel (because SDS is negatively charged) proportionally to their mass-to-charge ratio. As previously explained, this ratio will be nearly the same for all proteins. The proteins' progress will be slowed by frictional forces. The gel therefore acts like a molecular sieve when the current is applied, separating the proteins on the basis of their molecular weight with larger proteins being retained higher in the gel and smaller proteins being able to pass through the sieve and reach lower regions of the gel.

Detecting proteins

The result of this is a gel with proteins spread out on its surface. These proteins can then be detected by a variety of means, but the most commonly used stains are silver and Coomassie brilliant blue staining. In the former case, a silver colloid is applied to the gel. The silver binds to cysteine groups within the protein. The silver is darkened by exposure to ultra-violet light. The amount of silver can be related to the darkness, and therefore the amount of protein at a given location on the gel. This measurement can only give approximate amounts, but is adequate for most purposes. Silver staining is 100x more sensitive than Coomassie brilliant blue with a 40-fold range of linearity.[3]

Molecules other than proteins can be separated by 2D electrophoresis. In supercoiling assays, coiled DNA is separated in the first dimension and denatured by a DNA intercalator (such as ethidium bromide or the less carcinogenic chloroquine) in the second. This is comparable to the combination of native PAGE /SDS-PAGE in protein separation.

Common techniques

IPG-DALT

A common technique is to use an Immobilized pH gradient (IPG) in the first dimension. This technique is referred to as IPG-DALT. The sample is first separated onto IPG gel (which is commercially available) then the gel is cut into slices for each sample which is then equilibrated in SDS-mercaptoethanol and applied to an SDS-PAGE gel for resolution in the second dimension. Typically IPG-DALT is not used for quantification of proteins due to the loss of low molecular weight components during the transfer to the SDS-PAGE gel.[4]

IEF SDS-PAGE

See Isoelectric focusing

2D gel analysis software

Warping: Images of two 2D electrophoresis gels, overlaid with Delta2D. First image is colored in orange, second one colored in blue. Due to running differences, corresponding spots do not overlap.

Warping: Images of two 2D electrophoresis gels after warping. First image is colored in orange, second one colored in blue. Corresponding spots overlap after warping. Common spots are colored black, orange spots are only present (or much stronger) on the first image, blue spots are only present (or much stronger) on the second image.

In quantitative proteomics, these tools primarily analyze bio-markers by quantifying individual proteins, and showing the separation between one or more protein "spots" on a scanned image of a 2-DE gel. Additionally, these tools match spots between gels of similar samples to show, for example, proteomic differences between early and advanced stages of an illness. Software packages include Delta2D, ImageMaster, Melanie, PDQuest, Progenesis and REDFIN – among others.[citation needed] While this technology is widely utilized, the intelligence has not been perfected. For example, while PDQuest and Progenesis tend to agree on the quantification and analysis of well-defined well-separated protein spots, they deliver different results and analysis tendencies with less-defined less-separated spots.[5]

Challenges for automatic software-based analysis include incompletely separated (overlapping) spots (less-defined and/or separated), weak spots / noise (e.g., "ghost spots"), running differences between gels (e.g., protein migrates to different positions on different gels), unmatched/undetected spots, leading to missing values,[6][7] mismatched spots , errors in quantification (several distinct spots may be erroneously detected as a single spot by the software and/or parts of a spot may be excluded from quantification), and differences in software algorithms and therefore analysis tendencies

Generated picking lists can be used for the automated in-gel digestion of protein spots, and subsequent identification of the proteins by mass spectrometry. Mass spectrometry analysis can identify precise mass measurements along with the sequencing of peptides that range from 1000-4000 atomic mass units. [8] For an overview of the current approach for software analysis of 2DE gel images see[9] or.[10]

See also

• Difference gel electrophoresis
• QPNC-PAGE
• PROTOMAP

References

1. ^ O'Farrell, PH (1975). "High resolution two-dimensional electrophoresis of proteins". J. Biol. Chem. 250 (10): 4007–21. doi:10.1016/S0021-9258(19)41496-8. PMC 2874754. PMID 236308.
2. ^ Klose, J (1975). "Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals". Humangenetik. 26 (3): 231–43. doi:10.1007/bf00281458. PMID 1093965. S2CID 30981877.
3. ^ Switzer RC 3rd, Merril CR, Shifrin S (1979). "A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels". Analytical Biochemistry. 98 (1): 231–37. doi:10.1016/0003-2697(79)90732-2. PMID 94518.{{cite journal}}: CS1 maint: multiple names: authors list (link)
4. ^ Mikkelsen, Susan; Cortón, Eduardo (2004). Bioanalytical Chemistry. John Wiley & Sons, Inc. p. 224. ISBN 978-0-471-62386-1.
5. ^ Arora PS, Yamagiwa H, Srivastava A, Bolander ME, Sarkar G (2005). "Comparative evaluation of two two-dimensional gel electrophoresis image analysis software applications using synovial fluids from patients with joint disease". J Orthop Sci. 10 (2): 160–66. doi:10.1007/s00776-004-0878-0. PMID 15815863. S2CID 45193214.
6. ^ Pedreschi R, Hertog ML, Carpentier SC, et al. (April 2008). "Treatment of missing values for multivariate statistical analysis of gel-based proteomics data". Proteomics. 8 (7): 1371–83. doi:10.1002/pmic.200700975. hdl:1942/8262. PMID 18383008. S2CID 21152053.
7. ^ What are missing values, and why are they a problem?
8. ^ Lepedda, Antonio J, and Marilena Formato. “Applications of Two-Dimensional Electrophoresis Technology to the Study of Atherosclerosis.” EJIFCC vol. 19,3 146-59. 20 Dec. 2008
9. ^ Berth M, Moser FM, Kolbe M, Bernhardt J (October 2007). "The state of the art in the analysis of two-dimensional gel electrophoresis images". Appl. Microbiol. Biotechnol. 76 (6): 1223–43. doi:10.1007/s00253-007-1128-0. PMC 2279157. PMID 17713763.
10. ^ Bandow JE, Baker JD, Berth M, et al. (August 2008). "Improved image analysis workflow for 2-D gels enables large-scale 2-D gel-based proteomics studies--COPD biomarker discovery study". Proteomics. 8 (15): 3030–41. doi:10.1002/pmic.200701184. PMID 18618493. S2CID 206361897.

External links

Library resources about Two-dimensional gel electrophoresis

Resources in your library Resources in other libraries

• JVirGel Create virtual 2-D Gels from sequence data.
• Gel IQ A freely downloadable software tool for assessing the quality of 2D gel image analysis data.
• 2-D Electrophoresis Principles & Methods Handbook

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