Gel electrophoresis is a method for the separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, by size and cargo. It is used in clinical chemistry to separate proteins by charge and/or size (agarose IEF, essentially independent size) and in biochemistry and molecular biology to separate mixtures of DNA populations and RNA fragments by length, to estimate the size of DNA and RNA. fragment or separate the protein with the charge.
The nucleic acid molecule is separated by applying an electric field to move the negatively charged molecule through a matrix of agarose or other substance. The shorter molecules move faster and migrate farther than the longer because the shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sifting. Proteins are separated by a charge in agarose because the pores of the gel are too large to filter out the protein. Gel electrophoresis can also be used for the separation of nanoparticles.
The gel electrophoresis uses gel as an antifigtive and/or sieving medium during electrophoresis, the movement of charged particles in the electric field. The gel suppresses thermal convection caused by the application of an electric field, and can also act as a sieving medium, slowing down the molecular pathway; the gel also can only serve to keep the completed separation, so that the post electrophoresis stain can be applied. Gel gel electrophoresis is usually performed for analytical purposes, often after DNA amplification via polymerase chain reaction (PCR), but can be used as a preparative technique before using other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for more characterization continue.
Video Gel electrophoresis
Physical base
Simply put, electrophoresis is a process that allows the sorting of molecules by size. Using an electric field, molecules (such as DNA) can be made to move through gel made of agarose or polyacrylamide. The electric field consists of a negative charge at one end which drives molecules through the gel, and a positive charge at the other end that attracts molecules through the gel. The sorted molecules are thrown into the well in the gel material. The gel is placed in the electrophoresis chamber, which is then connected to a power source. When an electric current is applied, larger molecules move more slowly through the gel while smaller molecules move faster. Different sized molecules form different bands on the gel.
The term "gel" in this example refers to the matrix used to conceive, then separating the target molecule. In most cases, the gel is a cross polymer whose compositions and porosity are selected based on the weight and specific composition of the target to be analyzed. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel usually comprises different acrylamide concentrations and crosslinks, resulting in a mesh of polyacrylamide tissue of different sizes. When separating larger nucleic acids (more than a few hundred bases), the preferred matrix is ​​purified agarose. In both cases, the gel forms a dense, but porous matrix. Acrylamide, in contrast to polyacrylamide, is neurotoxin and should be treated using appropriate precautions to avoid toxicity. Agarose consists of long unbranched chains of uncharged carbohydrates without crosslinks that produce gel with large pores that allow for the separation of macromolecules and macromolecular complexes.
Electrophoresis refers to the electromotive force (EMF) used to move molecules through a gel matrix. By placing molecules in a well in a gel and applying an electric field, the molecules will move through the matrix at different levels, determined largely by their mass when the charge-to-mass ratio (Z) of all species is uniform. However, when the load is not all uniform, the electric field generated by the electrophoresis procedure will affect the species that have different charges and will therefore attract the species according to their alleged charges. Positively charged species will migrate towards the negatively charged cathode (since this is an electrolytic cell rather than galvanic). If the species is negatively charged they will migrate toward positively charged anodes.
If multiple samples have been loaded onto adjacent wells in the gel, they will run parallel on the individual path. Depending on the number of different molecules, each path shows the separation of components from the original mix as one or more distinct bands, one band per component. Incomplete component separation can lead to overlapping bands, or indistinguishable smears that represent some unresolved components. Bands on different paths ending at equal distances from above contain molecules passing through the gel at the same rate, which usually means they are roughly the same size. There is a marker of molecular weight sizes available containing a known molecular size mixture. If such markers are run on one track in a parallel gel with an unknown sample, the observed bands can be compared with the unknown to determine their size. The distance a band travels is inversely proportional to the logarithm of the molecular size.
There are limits to electrophoretic techniques. Because passing the current through the gel causes heating, the gel may melt during electrophoresis. Electrophoresis is carried out in a buffer solution to reduce the pH change due to the electric field, which is important because the DNA and RNA charge depend on pH, but running too long can deplete the buffering capacity of the solution. There is also a limit in determining molecular weight with SDS-PAGE, especially if you are trying to find MW from unknown proteins. There are certain biological variables that are difficult or impossible to minimize and may affect electrophoretic migration. These factors include protein structure, post-translational modification, and amino acid composition. For example, tropomyosin is an abnormal migrating acid protein in an SDS-PAGE gel. This is because acid residues are rejected by negatively charged SDS, leading to mass-to-charge and inaccurate migration ratios. Furthermore, different preparations of genetic material may not migrate consistently with each other, for morphological or other reasons.
Maps Gel electrophoresis
Gel type
The most commonly used gel types are agarose gel and polyacrylamide. Each gel type is suitable for different types and sizes of analyte. Polyacrylamide gel is usually used for proteins, and has very high breaking strength for small fragments of DNA (5-500 bp). Agarose gel on the other side has a lower cleavage for DNA but has a greater separation range, and is therefore used for DNA fragments typically 50-20,000 bp, but a resolution of more than 6 Mb is possible with pulsed field gel electrophoresis (PFGE ). ). The polyacrylamide gel is run in a vertical configuration while the agarose gel is usually run horizontally in submarine mode. They also differ in their casting methodologies, such as the agarose thermal set, while polyacrylamide is formed in chemical polymerization reactions.
Agarose
Agarose gel is made from natural polysaccharide polymer extracted from seaweed. The agarose gel is easily thrown and handled compared to other matrices, because the gel setting is a physical change rather than a chemical. Samples are also easy to recover. After the experiment is complete, the resulting gel can be stored in a plastic bag in the refrigerator.
Agarose gel does not have a uniform pore size, but is optimal for protein electrophoresis greater than 200 kDa. Agarose gel electrophoresis can also be used for separation of DNA fragments ranging from 50 base pairs to several megabases (millions of bases), the largest of which require specialized equipment. The distance between DNA bands of different lengths is influenced by the percent of agarose in the gel, with a higher percentage that takes longer, sometimes days. In contrast a high percentage agarose gel should be run with pulsed field electrophoresis (PFE), or inversion electrophoresis field.
"Most agarose gels are made with 0.7% (good separation or resolution of large 5-10kb DNA fragments) and 2% (good resolution for small fragments 0.2-1kb) agarose dissolved in electrophoretic buffers Up to 3% can be is used to separate very small fragments but the vertical polyacrylamide gel is more appropriate in this case, the low gel percentage is very weak and may break when you try to lift it, the high percentage of gel is often fragile and unevenly regulated, 1% gel is common for many applications. "
Polyacrylamide
Polyacrylamide gel electrophoresis (PAGE) is used to separate proteins with sizes ranging from 5 to 2,000 kDa due to uniform pore size provided by polyacrylamide gels. The pore size is controlled by modulating the acrylamide concentration and the bis-acrylamide powder used in gel preparation. Treatment should be used when making this type of gel, because acrylamide is a strong neurotoxin in the form of liquid and powder.
Traditional DNA sequencing techniques such as the Maxam-Gilbert or Sanger method employ polyacrylamide gels to separate different DNA fragments by a single long pair of bases so that the sequence can be read. Most modern DNA separation methods now use agarose gel, except for small DNA fragments. Currently most used in the field of immunological and protein analysis, it is often used to separate different proteins or isoforms from the same protein into separate bands. These can be transferred to the nitrocellulose membrane or PVDF to be examined with appropriate antibodies and markers, such as the western blot.
Usually finish the gel made in 6%, 8%, 10%, 12% or 15%. The accumulation gel (5%) is poured over the breaking gel and the comb gel (which forms the well and determines the path at which the protein, sample buffer and ladder will be placed) is inserted. The percentage chosen depends on the size of the protein you want to identify or examine in the sample. The smaller the weight is known, the higher the percentage should be used. Changes in gel buffer systems can help to resolve proteins of a very small size.
Starch
Partially hydrolyzed potato starch makes other non-toxic media for protein electrophoresis. The gel is slightly more opaque than acrylamide or agarose. The un-denatured protein can be separated according to the load and size. They are visualized using Napthal Black or Amido Black coloration. The typical starch gel concentration is 5% to 10%.
gel condition
Denaturing
The denaturation gel is run under conditions that interfere with the natural structure of the analyte, causing it to be revealed to be a linear chain. Thus, the mobility of each macromolecule depends only on its linear length and its mass-to-charge ratio. Thus, the level of secondary, tertiary, and quaternary biomolecular structures is disrupted, leaving only the main structure to be analyzed.
Nucleic acid is often denatured by introducing urea in a buffer, while the protein is 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 the tertiary and quaternary structures, a method called PAGE reduction. The reduction 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 required to approximate the exact molecular weight of RNA. RNA is capable of forming more intramolecular interactions than DNA which can lead to changes in electrophoretic mobility. Urea, DMSO and glyoxal are the most common denaturation agents used to disrupt RNA structures. Initially, highly toxic methylmercury hydroxide is often used in denaturing RNA electrophoresis, but it may be the method of choice for multiple samples.
The gel electrophoresis of denaturation is used in DNA-based pattern methods and RNA electrophoretic gel gradient gel (TGGE) and gel electrophoresis gel denaturation (DGGE).
Native
The original gel is run under non-denaturation conditions, so the natural structure of the analyte is maintained. This allows complex physical sizes to be folded or assembled to affect mobility, allowing for the analysis of the four levels of biomolecular structures. For biological samples, detergent is only used if necessary to lyse the lipid membrane inside the cell. The fixed complex - for the most part - is related and folded because they will be in the cell. One drawback, however, is that the complex may not be separately clean or predictable, as it is difficult to predict how the shape and size of the molecule will affect its mobility. Addressing and solving this problem is the ultimate goal of original quantitative PAGES.
Unlike the denaturation method, the original gel electrophoresis does not use a charged denaturation agent. The separated molecules (usually proteins or nucleic acids) are therefore not only different in molecular mass and intrinsic charge, but also the cross-sectional area, and thus undergo different electrophoretic forces depending on the overall shape of the structure. For proteins, since they remain in their original state they can be visualized not only by common protein dye reagents but also by specific staining of enzymes.
An example of a specific experiment from an original gel electrophoresis application is to examine enzymatic activity to verify the presence of enzymes in the sample during protein purification. For example, for an alkali phosphatase protein, the dye solution is a mixture of 4-chloro-2-2methylbenzenediazonium salt with 3-fo-2-naphthalic acid-2'-4'-dimethyl aniline in Tris buffer. This stain is sold commercially as a kit for gel staining. If the protein is present, the reaction mechanism takes place in the following order: beginning with de-phosphorylation of 3-phospho-2-naphthoic acid-2'-4'-dimethyl aniline by alkaline phosphatase (water required for the reaction). The phosphate group is released and replaced by a group of alcohols from water. The electrophile 4- chloro-2-2 methylbenzenediazonium (Fast Red TR Diazonium salt) replaces the alcohol groups that make up the end product of the Azo Red dye. As the name implies, this is a last seen red reaction product. In an academic experiment of protein purification scholars, the gel usually runs alongside commercial purified samples to visualize the results and create confusion as to whether the purification is successful or not.
Genuine gel electrophoresis is usually used in proteomics and metallomics. However, the original PAGE is also used to scan genes (DNA) for unknown mutations such as single strand conformation polymorphisms.
Buffer
Buffers in gel electrophoresis are used to provide ion-carrying currents and maintain pH at a relatively constant value. These buffers have many ions in them, which are necessary for the transfer of electricity through them. Something like distilled water or benzene contains few ions, which are not ideal for use in electrophoresis. There are a number of buffers used for electrophoresis. Most commonly, for Tris/Acetate/EDTA (TAE) nucleic acids, Tris/Borate/EDTA (TBE). Many other buffers have been proposed, eg. lithium borate, which is hardly ever used, based on Pubmed citations (LB), electrical histidine iso, pk buffer matched goods, etc.; in many cases the intended reason is a lower current (less heat) and or a suitable ion mobility, leading to longer buffer life. Borate is problematic; Borate can polymerize, and/or interact with the cis diol as found in RNA. TAE has the lowest buffer capacity but provides the best resolution for larger DNA. This means lower voltage and more time, but better product. LB is relatively new and ineffective in completing fragments greater than 5 kbp; However, with low conductivity, much higher voltages can be used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis. As low as one difference of base pair size can be solved with 3% agarose gel with very low conductivity medium (1 mM lithium Borat).
Most SDS-PAGE protein separations were performed using a "discontinuous" (or DISC) buffer system that significantly improved the band's sharpness in the gel. During electrophoresis in the gel system disconnected, the ion gradient is formed in the early stages of electrophoresis which causes all the proteins to focus into one sharp band in a process called isotachophoresis. The separation of proteins by size is achieved at the bottom, "solving" of the gel. The breaking gel usually has a much smaller pore size, leading to a sieving effect that now determines the electrophoretic mobility of the protein.
Visualization
After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. DNA can be visualized using ethidium bromide which, when inserted into DNA, glows under ultraviolet light, while the protein can be visualized using a silver dye or Coomassie Brilliant Blue dye. Other methods can also be used to visualize the separation of mixed components on the gel. If the separated molecule contains radioactivity, for example in a DNA sequencing gel, an autoradiogram can be recorded from the gel. Photos can be taken from the gel, often using the Gel Gel system.
Downstream
After separation, additional separation methods may be used, such as isoelectric focusing or SDS-PAGE. The gel will be physically cut, and the protein complex is extracted from each part separately. Each extract can then be analyzed, such as with a peptide mass fingerprint or de novo peptide sequencing after in-digestion gel. It can provide a lot of information about the identity of proteins in the complex.
Apps
- Estimation of the size of DNA molecules after digestion of restriction enzymes, eg. in the mapping of cloned DNA restrictions.
- Analysis of PCR products, e.g. in the diagnosis of molecular genetics or genetic fingerprints
- The separation of genomic DNA is limited before the transfer of the South, or RNA before the North transfers.
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 gel imaging devices. Images are recorded with a computer-operated camera, and the intensity of the ribbon or destination is measured and compared to standards or markers loaded on the same gel. Measurements and analysis are mostly done with special software.
Depending on the type of analysis performed, other techniques are often applied simultaneously with gel electrophoresis results, which provide a variety of field-specific applications.
Nucleic acid
In the case of nucleic acid, the direction of migration, from negative to positive electrode, is due to the natural negative charge carried by their sugar-phosphate backbone.
The double-stranded DNA fragments naturally behave like long rods, thus migrating them through the gels relative to their size or, to cyclic fragments, the radius of their circles. Circular DNAs such as plasmids, however, may exhibit multiple bands, the speed of migration may depend on whether it is relaxed or supercoiled. Single-stranded DNA or RNA tends to fold into molecules with complex shapes and migrate through the gel in a complex way based on their tertiary structure. Therefore, agents that interfere with hydrogen bonds, such as sodium hydroxide or formamide, are used to change the nature of nucleic acids and cause them to behave like longer stems.
DNA gel or large RNA electrophoresis is usually performed with agarose gel electrophoresis. See page "Chain termination method" for examples of DNA sequencing polyacrylamide gel. Characterization through the interaction of nucleic acid ligand or fragments can be performed by electrophoresis of mobility shift affinity.
RNA sample electrophoresis can be used to check the genomic DNA contamination and also for RNA degradation. RNA from eukaryotic organisms shows different bands of rRNA 28 and rRNA 18s, the 28s band becomes approximately twice as strong as the 18s band. Degraded RNA has a less sharp band, has a dirty appearance, and an intensity ratio of less than 2: 1.
Protein
Proteins, unlike nucleic acids, can have varied charges and complex shapes, therefore they can not migrate to the polyacrylamide gel at the same level, or at all, when placing the negative EMF into the positive in the sample. Therefore, proteins are usually denatured in the presence of detergents such as sodium dodecyl sulphate (SDS) that coat proteins with negative charges. Generally, the amount of SDS is bound relative to the size of the protein (usually 1.4g SDS per gram of protein), so the resulting denatur protein has an overall negative charge, and all proteins have the same mass-to-mass. comparison. Since the denatured protein acts like a long rod rather than having a complex tertiary shape, the rate at which the resulting SDS-coated proteins migrate in the gel relative to its size and not its charge or shape.
Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), with original gel electrophoresis, with preparative gel electrophoresis (QPNC-PAGE), or with 2-D electrophoresis.
Characterization through ligand interaction can be performed with electroblotting or by affinity electrophoresis in agarose or with capillary electrophoresis such as for estimating binding constants and the determination of structural features such as glycan content via binding lectins.
History
- 1930 - first report on the use of sucrose for gel electrophoresis
- 1955 - introduction of starch gel, mediocre separation (Smithies)
- 1959 - introduction of acrylamide gel; disk electrophoresis (Ornstein and Davis); accurate parameter control such as pore size and stability; and (Raymond and Weintraub)
- 1966 - first use gel gel
- 1969 - introduction of denaturation agents especially SDS separation of protein subunit (Weber and Osborn)
- 1970 - Laemmli separates 28 T4 phage components using stacking gels and SDS
- 1972 - agarose gel with ethidium bromide staining
- 1975 - 2-D Gel (O'Farrell); isoelectric focus then SDS gel electrophoresis
- 1977 - gel sequencing
- 1983 - pulsed field gel electrophoresis allows separation of large DNA molecules
- 1983 - introduction of capillary electrophoresis
- 2004 - standard timing of acrylamide gel polymerization allows the clearance of clean and predictable native proteins (Kastenholz)
A 1959 book on electrophoresis by Milan Bier cites references from the 1800s. However, Oliver Smithies made a significant contribution. Bier states: "The Smithies method... finds wide application due to its unique separation forces." Taken in context, Bier clearly implies that Smithies' method is an improvement.
See also
References
External links
- Electrophoresis Demonstration of Biotechnical Laboratory, from Utah University of Genetics Science Center
- The original gel protein electrophoresis falters
- Drink straw electrophoresis
- How to run DNA or RNA gel
- Animated gel analysis from DNA restrictions
- Step-by-step photos to run gel and extract DNA
- A typical method of wikiversity
- Principle of Electrophoresis 2-D & amp; Handbook Method
Source of the article : Wikipedia
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