Gel electrophoresis - AbsoluteAstronomy.com

Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size (IEF agarose, essentially size independent) and in biochemistry

Biochemistry

Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...

and molecular biology

Molecular biology

Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

to separate a mixed population of DNA

DNA

Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

and RNA

RNA

Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

fragments by length, to estimate the size of DNA

DNA

and RNA

RNA

Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

fragments or to separate protein

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s by charge. Nucleic acid molecules are separated by applying an electric field

Electric field

In physics, an electric field surrounds electrically charged particles and time-varying magnetic fields. The electric field depicts the force exerted on other electrically charged objects by the electrically charged particle the field is surrounding...

to move the negatively charged molecules through an agarose matrix. 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. Proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins. Gel electrophoresis can also be used for separation of nanoparticles.

Gel electrophoresis uses a gel as an anticonvective medium and or sieving medium during electrophoresis, the movement of a charged particle in an electrical field. Gels suppress the thermal convection caused by application of the electric field, and can also act as a sieving medium, retarding the passage of molecules; gels can also simply serve to maintain the finished separation, so that a post electrophoresis stain can be applied. DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR, but may be used as a preparative technique prior to use of other methods such as mass spectrometry

Mass spectrometry

Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...

, RFLP

Restriction fragment length polymorphism

In molecular biology, restriction fragment length polymorphism, or RFLP , is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related...

, PCR

Polymerase chain reaction

The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....

, cloning

Cloning

Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...

, DNA sequencing

DNA sequencing

DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....

, or Southern blot

Southern blot

A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...

ting for further characterization.

Separation

In simple terms: Electrophoresis is a procedure which enables the sorting of molecules based on size and charge. Using an electric field, molecules (such as DNA) can be made to move through a gel made of agar

Agar

Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae. Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium...

or polyacrylamide

Polyacrylamide

Polyacrylamide is a polymer formed from acrylamide subunits. It can be synthesized as a simple linear-chain structure or cross-linked, typically using N,N-methylenebisacrylamide. Polyacrylamide is not toxic...

. 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 current 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.

The term "gel

Gel

A gel is a solid, jelly-like material that can have properties ranging from soft and weak to hard and tough. Gels are defined as a substantially dilute cross-linked system, which exhibits no flow when in the steady-state...

" in this instance refers to the matrix used to contain, then separate the target molecules. In most cases, the gel is a crosslinked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. When separating protein

Protein

s or small nucleic acid

Nucleic acid

Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...

s (DNA

DNA

, RNA

RNA

Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

, or oligonucleotide

Oligonucleotide

An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...

s) the gel is usually composed of different concentrations of acrylamide

Acrylamide

Acrylamide is a chemical compound with the chemical formula C3H5NO. Its IUPAC name is prop-2-enamide. It is a white odourless crystalline solid, soluble in water, ethanol, ether, and chloroform. Acrylamide is incompatible with acids, bases, oxidizing agents, iron, and iron salts...

and a cross-linker, producing different sized mesh networks of polyacrylamide

Polyacrylamide

. When separating larger nucleic acids (greater than a few hundred bases

Base (chemistry)

For the term in genetics, see base A base in chemistry is a substance that can accept hydrogen ions or more generally, donate electron pairs. A soluble base is referred to as an alkali if it contains and releases hydroxide ions quantitatively...

), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix. Acrylamide

Acrylamide

, in contrast to polyacrylamide

Polyacrylamide

, is a neurotoxin

Neurotoxin

A neurotoxin is a toxin that acts specifically on nerve cells , usually by interacting with membrane proteins such as ion channels. Some sources are more general, and define the effect of neurotoxins as occurring at nerve tissue...

and must be handled using appropriate safety precautions to avoid poisoning. Agarose is composed of long unbranched chains of uncharged carbohydrate without cross links resulting in a gel with large pores allowing for the separation of macromolecules and macromolecular complexes

Affinity electrophoresis

Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. The methods include the so-called mobility shift electrophoresis, charge shift...

.

"Electrophoresis

Electrophoresis

Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...

" refers to the electromotive force

Electromotive force

In physics, electromotive force, emf , or electromotance refers to voltage generated by a battery or by the magnetic force according to Faraday's Law, which states that a time varying magnetic field will induce an electric current.It is important to note that the electromotive "force" is not a...

(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, toward the anode

Anode

An anode is an electrode through which electric current flows into a polarized electrical device. Mnemonic: ACID ....

if negatively charged or toward the cathode

Cathode

A cathode is an electrode through which electric current flows out of a polarized electrical device. Mnemonic: CCD .Cathode polarity is not always negative...

if positively charged.

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 separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components. Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are molecular weight size marker

Molecular weight size marker

A molecular weight size marker is used to identify the approximate size of a molecule run on a gel, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix...

s available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule.

There are limits to electrophoretic techniques. Since passing 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. Further, different preparations of genetic material may not migrate consistently with each other, for morphological or other reasons.

Agarose

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 gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair

Base pair

In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...

to several megabases (millions of bases) using specialized apparatus. The distance between DNA bands of a given length is determined by the percent agarose in the gel. The disadvantage of higher concentrations is the long run times (sometimes days). Instead high percentage agarose gels should be run with a pulsed field electrophoresis

Pulsed field gel electrophoresis

Pulsed field gel electrophoresis is a technique used for the separation of large deoxyribonucleic acid molecules by applying an electric field that periodically changes direction to a gel matrix.-Historical background:...

(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."

Agarose gels do not have a uniform pore size, but are optimal for electrophoresis of proteins that are larger than 200 kDa.

Polyacrylamide

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 controlling 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 form.

Traditional DNA sequencing

DNA sequencing

DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....

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

Immunology

Immunology is a broad branch of biomedical science that covers the study of all aspects of the immune system in all organisms. It deals with the physiological functioning of the immune system in states of both health and diseases; malfunctions of the immune system in immunological disorders ; the...

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

Nitrocellulose

Nitrocellulose is a highly flammable compound formed by nitrating cellulose through exposure to nitric acid or another powerful nitrating agent. When used as a propellant or low-order explosive, it is also known as guncotton...

or PVDF membrane to be probed with antibodies and corresponding markers, such as in a western blot

Western blot

The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...

.

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 on the buffer system of the gel can help to further resolve proteins of very small sizes.

Denaturing

A denaturing gel is a type of electrophoresis in which the native structure of macromolecule

Macromolecule

A macromolecule is a very large molecule commonly created by some form of polymerization. In biochemistry, the term is applied to the four conventional biopolymers , as well as non-polymeric molecules with large molecular mass such as macrocycles...

s that are run within the gel is not maintained. For instance, gels used in SDS-PAGE

SDS-PAGE

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related techniques widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility...

(sodium dodecyl sulfate

Sodium dodecyl sulfate

Sodium dodecyl sulfate , sodium laurilsulfate or sodium lauryl sulfate is an organic compound with the formula CH311OSO3Na). It is an anionic surfactant used in many cleaning and hygiene products...

polyacrylamide

Polyacrylamide

gel electrophoresis) will unfold and denature the native structure of a protein

Protein

. In contrast to native gel electrophoresis, quaternary structure

Quaternary structure

In biochemistry, quaternary structure is the arrangement of multiple folded protein or coiling protein molecules in a multi-subunit complex.-Description and examples:...

cannot be investigated using this method.

Native

Native gel electrophoresis is an electrophoretic separation method typically used in proteomics

Proteomics

Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...

and metallomics

Metallome

The term metallome has been introduced by R.J.P. Williams by analogy with proteome as distribution of free metal ions in every one of cellular compartments. Subsequently, the term metallomics has been coined as the study of metallome. Szpunar defined metallomics as "comprehensive analysis of the...

.

Native PAGE separations are run in non-denaturing conditions. Detergents are used only to the extent that they are necessary to lyse

Lysis

Lysis refers to the breaking down of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a "lysate"....

lipid membranes

Lipid bilayer

The lipid bilayer is a thin membrane made of two layers of lipid molecules. These membranes are flat sheets that form a continuous barrier around cells. The cell membrane of almost all living organisms and many viruses are made of a lipid bilayer, as are the membranes surrounding the cell nucleus...

in the cell

Cell (biology)

The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....

. 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, since they cannot move through the polyacrylamide gel as quickly as individual, denatured proteins.

Unlike denaturing methods, such as SDS-PAGE

SDS-PAGE

, native gel electrophoresis does not use a charged denaturing

Denaturation (biochemistry)

Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...

agent. The molecules being separated (usually proteins or nucleic acids) therefore differ not only in molecular mass

Molecular mass

The molecular mass of a substance is the mass of one molecule of that substance, in unified atomic mass unit u...

and intrinsic charge, but also the cross-sectional area, and thus experience different electrophoretic forces dependent on the shape of the overall structure. Since the proteins remain in the native state they may be visualised not only by general protein staining reagents but also by specific enzyme-linked staining.

Buffers

There are a number of buffers used for electrophoresis. The most common being, for nucleic acids Tris/Acetate/EDTA

TAE buffer

TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA.In molecular biology it is used in agarose electrophoresis typically for the separation of nucleic acids such as DNA and RNA. It is made up of Tris-acetate buffer, usually at pH 8.0, and EDTA, which sequesters...

(TAE), Tris/Borate/EDTA

TBE Buffer

TBE or Tris/Borate/EDTA, is a buffer solution containing a mixture of Tris base, boric acid and EDTA.In molecular biology, TBE and TAE buffers are often used in procedures involving nucleic acids, the most common being electrophoresis. Tris-acid solutions are effective buffers for slightly basic...

(TBE). Many other buffers have been proposed, e.g. lithium borate

LB buffer

Lithium Borate buffer is a buffer solution used in agarose electrophoresis, typically for the separation of nucleic acids such as DNA and RNA. It is made up of Lithium borate ....

, which is almost never used, based on Pubmed citations (LB), iso electric histidine, pK matched goods buffers, etc.; in most cases the purported rationale is lower current (less heat) and or matched ion mobilities, which leads to longer buffer life. Borate is problematic; Borate can polymerize, and/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).

Visualization

After the electrophoresis is complete, the molecules in the gel can be stained

Staining (biology)

Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes...

to make them visible. DNA may be visualized using ethidium bromide

Ethidium bromide

Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane...

which, when intercalated into DNA, fluoresce

Fluorescence

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...

under ultraviolet

Ultraviolet

Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...

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 DNA sequencing

DNA sequencing

DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....

gel, an autoradiogram can be recorded of the gel. Photograph

Photograph

A photograph is an image created by light falling on a light-sensitive surface, usually photographic film or an electronic imager such as a CCD or a CMOS chip. Most photographs are created using a camera, which uses a lens to focus the scene's visible wavelengths of light into a reproduction of...

s can be taken of gels, often using Gel Doc

Gel Doc

Gel Doc, also known as Gel Documentation System, Gel Image System or Gel Imager, is widely used in molecular biology laboratories for the imaging and documentation of nucleic acid and protein polyacrylamide or agarose gels typically stained with ethidium bromide or other fluorophores such as SYBR...

.

The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis is ethidium bromide

Ethidium bromide

, usually abbreviated as EtBr. It fluoresces under UV light when intercalated into the major groove of DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with UV light, any band containing more than ~20 ng DNA becomes distinctly visible. EtBr is a known mutagen, and safer alternatives are available, such as GelRed, which binds to the minor groove.

SYBR Green I

SYBR Green

SYBR Green I is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to DNA. The resulting DNA-dye-complex absorbs blue light and emits green light . The stain preferentially binds to double-stranded DNA, but will stain single-stranded DNA with lower...

is another dsDNA stain, produced by Invitrogen

Invitrogen

Invitrogen Corporation was a large, multinational biotechnology company headquartered in Carlsbad, California. In November 2008, a merger between Applied Biosystems and Invitrogen was finalized...

. It is more expensive, but 25 times more sensitive, and possibly safer than EtBr, though there is no data addressing its mutagenicity or toxicity in humans.

SYBR Safe is a variant of SYBR Green that has been shown to have low enough levels of mutagenicity and toxicity to be deemed nonhazardous waste under U.S. Federal regulations. It has similar sensitivity levels to EtBr, but, like SYBR Green, is significantly more expensive. In countries where safe disposal of hazardous waste is mandatory, the costs of EtBr disposal can easily outstrip the initial price difference, however.

Since EtBr stained DNA is not visible in natural light, scientists mix DNA with negatively charged loading buffers before adding the mixture to the gel. Loading buffers are useful because they are visible in natural light (as opposed to UV light for EtBr stained DNA), and they co-sediment with DNA (meaning they move at the same speed as DNA of a certain length). Xylene cyanol

Xylene cyanol

Xylene cyanol can be used as a colour marker to monitor the process of agarose gel electrophoresis and polyacrylamide gel electrophoresis. Bromophenol blue and orange G can also be used for this purpose.-Migration speed:...

and Bromophenol blue

Bromophenol blue

Bromophenol blue is used as an acid-base indicator, a color marker and a dye.-Acid-base indicator:As an acid-base indicator its useful range lies between pH 3.0 and 4.6...

are common dyes found in loading buffers; they run about the same speed as DNA fragments that are 5000 bp and 300 bp in length respectively, but the precise position varies with percentage of the gel. Other less frequently used progress markers are Cresol Red

Cresol Red

Cresol Red is a triarylmethane dye frequently used for monitoring the pH in aquaria.-pH indicator:-Molecular biology:...

and Orange G

Orange G

Orange G or orange gelb is a synthetic azo dye used in histology in many staining formulations. It usually comes as a disodium salt. It has the appearance of orange crystals or powder.-Staining:...

which run at about 125 bp and 50 bp, respectively.

Visualization can also be achieved by transferring DNA to a nitrocellulose membrane followed by exposure to a hybridization probe

Hybridization probe

In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length , which is used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe...

. This process is termed Southern blot

Southern blot

ting.

Analysis

After electrophoresis the gel is illuminated with an ultraviolet

Ultraviolet

Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...

lamp (usually by placing it on a light box, while using protective gear to limit exposure to ultraviolet radiation). The illuminator apparatus mostly also contains imaging apparatus that takes an image of the gel, after illumination with UV radiation. The ethidium bromide

Ethidium bromide

fluoresces

Fluorescence

reddish-orange in the presence of DNA, since it has intercalated with the DNA. The DNA band can also be cut out of the gel, and can then be dissolved to retrieve the purified DNA.
The gel can then be photographed usually with a digital or polaroid camera. Although the stained nucleic acid fluoresces reddish-orange, images are usually shown in black and white (see figures).

Even short exposure of nucleic acids to UV light causes significant damage to the sample. UV damage to the sample will reduce the efficiency of subsequent manipulation of the sample, such as ligation and cloning. If the DNA is to be used after separation on the agarose gel, it is best to avoid exposure to UV light by using a blue light excitation source such as the XcitaBlue UV to blue light conversion screen from Bio-Rad

Bio-Rad

Bio-Rad Laboratories, Inc. , was founded in 1952 in Berkeley, California. The company was initially engaged in the development and production of specialty chemicals used in biochemical, pharmaceutical, and other life science research applications...

or Dark Reader from Clare Chemicals. A blue excitable stain is required, such as one of the SYBR Green or GelGreen stains. Blue light is also better for visualization since it is safer than UV (eye-protection is not such a critical requirement) and passes through transparent plastic and glass. This means that the staining will be brighter even if the excitation light goes through glass or plastic gel platforms.

Gel electrophoresis research often takes advantage of software-based image analysis tools, such as ImageJ

ImageJ

ImageJ is a public domain, Java-based image processing program developed at the National Institutes of Health. ImageJ was designed with an open architecture that provides extensibility via Java plugins and recordable macros. Custom acquisition, analysis and processing plugins can be developed using...

1	2	3

Downstream processing

After separation, an additional separation method may then be used, such as isoelectric focusing

Isoelectric focusing

Isoelectric focusing , also known as electrofocusing, is a technique for separating different molecules by their electric charge differences...

or SDS-PAGE

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

Peptide mass fingerprinting

Peptide mass fingerprinting is an analytical technique for protein identification that was developed in 1993 by several groups independently. In this method, the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass...

or de novo sequencing after in-gel digestion

In-gel digestion

The in-gel digestion is part of the sample preparation for the mass spectrometric identification of proteins in course of proteomic analysis. The method was introduced 1992 by Rosenfeld...

. 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
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...

of cloned DNA.
Analysis of PCR products, e.g. in molecular genetic diagnosis
Preimplantation genetic diagnosis
In medicine and genetics pre-implantation genetic diagnosis refers to procedures that are performed on embryos prior to implantation, sometimes even on oocytes prior to fertilization. PGD is considered another way to prenatal diagnosis...

or genetic fingerprinting
Genetic fingerprinting
DNA profiling is a technique employed by forensic scientists to assist in the identification of individuals by their respective DNA profiles. DNA profiles are encrypted sets of numbers that reflect a person's DNA makeup, which can also be used as the person's identifier...
Separation of restricted genomic DNA prior to Southern transfer
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...

, or of RNA prior to Northern transfer
Northern blot
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation,...

.

Gel electrophoresis is used in forensics

Forensic chemistry

Forensic chemistry is the application of chemistry to law enforcement or the failure of products or processes. Many different analytical methods may be used to reveal what chemical changes occurred during an incident, and so help reconstruct the sequence of events...

, molecular biology

Molecular biology

, genetics

Genetics

Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....

, microbiology

Microbiology

Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell , cell clusters or no cell at all . This includes eukaryotes, such as fungi and protists, and prokaryotes...

and biochemistry

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

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

Sugar

Sugar is a class of edible crystalline carbohydrates, mainly sucrose, lactose, and fructose, characterized by a sweet flavor.Sucrose in its refined form primarily comes from sugar cane and sugar beet...

-phosphate

Phosphate

A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...

backbone.

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

Radius of gyration

Radius of gyration or gyradius is the name of several related measures of the size of an object, a surface, or an ensemble of points. It is calculated as the root mean square distance of the objects' parts from either its center of gravity or an axis....

. Circular DNA such as plasmid

Plasmid

In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...

s, however, may show multiple bands, the speed of migrate may depend on whether it is relaxed or supercoiled. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bond

Hydrogen bond

A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. The hydrogen must be covalently bonded to another electronegative atom to create the bond...

s, such as sodium hydroxide or formamide

Formamide

Formamide, also known as methanamide, is an amide derived from formic acid. It is a clear liquid which is miscible with water and has an ammonia-like odor. It is used primarily for manufacturing sulfa drugs and synthesizing vitamins and as a softener for paper and fiber...

, are used to denature the nucleic acids and cause them to behave as long rods again.

Gel electrophoresis of large DNA

DNA

or RNA

RNA

Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

is usually done by agarose gel electrophoresis. See the "Chain termination method" page for an example of a polyacrylamide

Polyacrylamide

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

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 sharpely defined bands, has a smeared appearance, and intensity ratio is less than 2:1.

Proteins

Protein

s, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the polyacrylamide gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are usually denatured

Denaturation (biochemistry)

in the presence of a detergent

Detergent

A detergent is a surfactant or a mixture of surfactants with "cleaning properties in dilute solutions." In common usage, "detergent" refers to alkylbenzenesulfonates, a family of compounds that are similar to soap but are less affected by hard water...

such as sodium dodecyl sulfate

Sodium dodecyl sulfate

Sodium dodecyl sulfate , sodium laurilsulfate or sodium lauryl sulfate is an organic compound with the formula CH311OSO3Na). It is an anionic surfactant used in many cleaning and hygiene products...

/sodium dodecyl phosphate (SDS/SDP) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per gram of protein), so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape.

Protein

Protein

s are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE

SDS-PAGE

), by native gel electrophoresis, by quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE

QPNC-PAGE

QPNC-PAGE, or quantitative preparative native continuous polyacrylamide gel electrophoresis, is a high-resolution technique applied in biochemistry and bioinorganic chemistry to separate proteins by isoelectric point...

), or by 2-D electrophoresis.

Characterization through ligand interaction may be performed by electroblotting

Electroblotting

Electroblotting is a method in molecular biology/biochemistry/immunogenetics to transfer proteins or nucleic acids onto a membrane by using PVDF or nitrocellulose, after gel electrophoresis. The protein or nucleic acid can then be further analyzed using probes such as specific antibodies, ligands...

or by affinity electrophoresis

Affinity electrophoresis

in agarose or by capillary electrophoresis

Capillary electrophoresis

Capillary electrophoresis , also known as capillary zone electrophoresis , can be used to separate ionic species by their charge and frictional forces and hydrodynamic radius. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an...

as for estimation of binding constants and determination of structural features like glycan content through lectin

Lectin

Lectins are sugar-binding proteins that are highly specific for their sugar moieties. They play a role in biological recognition phenomena involving cells and proteins. For example, some viruses use lectins to attach themselves to the cells of the host organism during infection...

binding.

History

1930s – first reports of the use of sucrose
Sucrose
Sucrose is the organic compound commonly known as table sugar and sometimes called saccharose. A white, odorless, crystalline powder with a sweet taste, it is best known for its role in human nutrition. The molecule is a disaccharide composed of glucose and fructose with the molecular formula...

for gel electrophoresis
1955 – introduction of starch
Starch
Starch or amylum is a carbohydrate consisting of a large number of glucose units joined together by glycosidic bonds. This polysaccharide is produced by all green plants as an energy store...

gels, mediocre separation
1959 – introduction of acrylamide
Acrylamide
Acrylamide is a chemical compound with the chemical formula C3H5NO. Its IUPAC name is prop-2-enamide. It is a white odourless crystalline solid, soluble in water, ethanol, ether, and chloroform. Acrylamide is incompatible with acids, bases, oxidizing agents, iron, and iron salts...

gels; disc electrophoresis (Ornstein and Davis); accurate control of parameters such as pore size and stability; and (Raymond and Weintraub)
1969 – introduction of denaturing
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...

agents especially SDS
Sodium dodecyl sulfate
Sodium dodecyl sulfate , sodium laurilsulfate or sodium lauryl sulfate is an organic compound with the formula CH311OSO3Na). It is an anionic surfactant used in many cleaning and hygiene products...

separation of protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

subunit (Weber and Osborn)
1970 – Laemmli separated 28 components of T4 phage using a stacking gel and SDS
1975 – 2-dimensional gels (O’Farrell); isoelectric focusing
Isoelectric focusing
Isoelectric focusing , also known as electrofocusing, is a technique for separating different molecules by their electric charge differences...

then SDS gel electrophoresis
1977 – sequencing
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....

gels
late 1970s – agarose gels
1983 – pulsed field gel electrophoresis
Pulsed field gel electrophoresis
Pulsed field gel electrophoresis is a technique used for the separation of large deoxyribonucleic acid molecules by applying an electric field that periodically changes direction to a gel matrix.-Historical background:...

enables separation of large DNA molecules
1983 – introduction of capillary electrophoresis
Capillary electrophoresis
Capillary electrophoresis , also known as capillary zone electrophoresis , can be used to separate ionic species by their charge and frictional forces and hydrodynamic radius. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an...

A 1959 book on electrophoresis by Milan Bier cites references from the 1800s. However, Oliver Smithies

Oliver Smithies

Oliver Smithies is a British-born American geneticist and Nobel laureate, credited with the invention of gel electrophoresis in 1955, and the simultaneous discovery, with Mario Capecchi and Martin Evans, of the technique of homologous recombination of transgenic DNA with genomic DNA, a much more...

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.

External links

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 fragments
Detailed description and movies of the preparation and uses of agarose gels
Step by step photos of running a gel and extracting DNA
Drinking straw electrophoresis!
A typical method from wikiversity

The source of this article is wikipedia, the free encyclopedia. The text of this article is licensed under the GFDL.

Separation

Agarose

Polyacrylamide

Denaturing

Native

Buffers

Visualization

Analysis

Downstream processing

Applications

Nucleic acids

Proteins

History

See also

External links