SDS-PAGE ( sodium dodecyl sulfate-polyacrylamide gel electrophoresis ) is a variant of polyacrylamide gel electrophoresis, an analytical method in biochemistry for the separation of charged molecules in the mixture by their molecules. mass in the electric field. It uses sodium dodecyl sulfate molecules (SDS) to help identify and isolate protein molecules.
The discontinuous electrophoretic system developed by Ulrich K. Laemmli enables good separation of proteins with molecular masses between 5 and 250 KDa. The publications that explain it are the papers most frequently cited by a single author, and the second most cited as a whole.
Video SDS-PAGE
Properti
SDS-PAGE is a method of electrophoresis that allows the separation of proteins by mass. Medium (also referred to as? Matrix?) Is a polyacrylamide-based disconnected gel. In addition, SDS (sodium dodecyl sulfate) is used. About 1.4 grams of SDS binds to one gram of protein, corresponding to one molecule of SDS per two amino acids. SDS acts as a surfactant, masking the intrinsic charge of proteins and giving them a very similar charge-to-mass ratio. The intrinsic charge of the protein is negligible compared to the loading of SDS, and the positive charge is also greatly reduced in the basic pH range of the separating gel. After the application of a constant electric field, the proteins migrate toward the anode, each at different speeds, depending on their mass. This simple procedure allows the proper separation of proteins by mass.
SDS tends to form spherical micelles in an aqueous solution over a certain concentration called critical micelle concentration (CMC). Above the critical critical mass of 7 to 10 millimolar in solution, SDS simultaneously occurs as a single molecule (monomer) and as micelles, under CMC SDS occurs only as a monomer in an aqueous solution. At critical micelle concentrations, micelles comprise about 62 molecules of SDS. However, only SDS monomers bind proteins through hydrophobic interactions, whereas the SDS micelles are anionic on the outside and do not absorb any protein. SDS is amphipathic, allowing it to open up parts of the structure of polar and nonpolar proteins. In SDS concentrations above 0.1 millimolar, protein opening begins, and above 1 mM, most of the proteins are denatured. Due to the strong denaturation effect of SDS and the subsequent dissociation of protein complexes, quartener structures generally can not be determined with SDS. Exception is e.g. proteins previously stabilized by covalent crosslinks and SDS-resistant protein complexes, which are stable even in the presence of SDS (the latter, however, only at room temperature). To change the complex properties of resistant SDS required high activation energy, which is achieved by heating. SDS Resistance is based on the metastability of protein folds. Although genuine, fully folded, the SDS-resistant protein does not have sufficient stability in the presence of SDS, the chemical equilibrium denaturation at room temperature occurs slowly. The stable protein complex is not only characterized by SDS resistance but also by the stability of the protease and the increase in biological half-life.
Alternatively, polyacrylamide gel electrophoresis may also be performed with a CTAB cationic surfactant in CTAB-PAGE, or 16-BAC in BAC-PAGE.
Maps SDS-PAGE
Procedures
The SDS-PAGE method consists of gel preparation, sample preparation, electrophoresis, protein staining or western blotting and the resulting ribbon pattern analysis.
Gel production
When using different buffers in the gel (gel electrophoresis intermittently), the gel is made up to one day before electrophoresis, so the diffusion does not lead to mixing the buffer. This gel is produced by radical polymerization in a mold consisting of two glass plates closed with a spacer between glass plates. In a typical mini-gel arrangement, the spacer has a thickness of 0.75 mm or 1.5 mm, which determines the gel loading capacity. To pour the gel solution, the plates are usually clamped into a stand that temporarily covers the bottom of the glass plate with two spacers. For a gel solution, acrylamide is mixed as a former gel (usually 4% V/V in a stacking gel and 10-12% in a separating gel), methylenebisacrylamide as a cross-linker, stacker or gel separation, water and SDS.. By adding TEMED catalysts and initiators of ammonium persulfate radical (APS) polymerization begins. The solution is then poured in between glass plates without making bubbles. Depending on the number of catalysts and radical starters and depending on temperature, the polymerization lasts between a quarter of an hour and several hours. The lower gel (gel separator) is poured first and covered with a few drops of alcohol that is almost insoluble in water (usually buffer-saturated butanol or isopropanol), which removes the bubbles from the meniscus and protects the radical scavenger oxygen gel solution. After gel separation polymerization, the alcohol is discarded and the residual alcohol removed by the filter paper. After the addition of APS and TEMED to a stacking gel solution, it is poured over a solid separation gel. After that, an appropriate comb comb is inserted between the glass plate without creating a bubble. The sample comb is carefully pulled after polymerization, leaving the bag for the sample application. For subsequent use of proteins for protein sequencing, gels are often prepared a day before electrophoresis to reduce the reaction of acrylamide that is not polymerized with cysteine ​​in the protein.
Using a gradient mixer, gradient gel with acrylamide gradient (usually from 4 to 12%) can be cast, which has a greater molecular mass separation range. Commercial gel systems (commonly called pre-printed gels) typically use a methane bis-tris buffer material with a pH value between 6.4 and 7.2 in both the stacking gel and in the separating gel. This gel is cast and ready for use. Because they only use one buffer (continuous gel electrophoresis) and have a nearly neutral pH, they can be stored for several weeks. The more neutral PH slows the hydrolysis and thus the decomposition of the polyacrylamide. In addition, there are fewer modified cysteine ​​acrylamide in the protein. Due to the constant pH in collecting and separating the gel there is no stacking effect. Proteins in BisTris gel can not be colored with ruthenium complexes. This gel system has a relatively large separation range, which can be varied by using MES or MOPS in a running buffer.
Sample preparation
During sample preparation, the sample buffer, and thus SDS, is added excessively to the protein, and the sample is then heated to 95 ° C for five minutes to disrupt the secondary and tertiary structures by disrupting the hydrogen bonds and stretching the molecules. Optionally, the disulphide bridge can be cleaved by reduction. For this purpose, reducing thiols like? -daptaptanol (? -Me, 5% volume), dithiothreitol (DTT, 10 millimolar) or dithioerythritol (DTE, 10 millimolar) was added to the sample buffer. After cooling to room temperature, each sample is piped into its own pocket in a gel, previously soaked in an electrophoresis buffer in an electrophoresis device.
In addition to samples, the molecular-weight size marker is usually loaded onto the gel. It consists of a known size protein and thus allows estimation (with errors Ã, Â ± 10%) of the size of the protein in the actual sample, which migrates in parallel on different tracks of the gel. Marker size is often piped into the first or last gel bag.
Electrophoresis
For separation, the denatured sample is loaded onto a polyacrylamide gel, which is placed in an electrophoretic buffer with an appropriate electrolyte. After that, a voltage (usually about 100 V, 10-20 V per cm of gel length) is applied, which causes the migration of negatively charged molecules through the gel toward the anode (pole plus). The gel acts like a sieve. Small proteins migrate relatively easily through the gel mesh, while larger proteins are more likely to be retained and thus migrate more slowly through the gel. Electrophoresis lasts between three quarters of an hour and several hours depending on the stress and length of the gel used.
The fastest migrating protein (with a molecular weight less than 5 KDa) forms the front buffer along with the anionic component of the electrophoresis buffer, which also migrates through the gel. The front buffer area is made visible by adding a relatively small blue anionic blue bromophenol blue to the sample buffer. Because of the relatively small size of the bromophenol blue molecule, it migrates faster than proteins. With optical controls of colored tapes migrating, electrophoresis can be stopped before dyeing and also the sample has actually migrated through the gel and leaving it.
The most commonly used method is the discontinuous SDS-PAGE. In this method, proteins migrate first into the collecting gel with a neutral pH, where they are concentrated and then they migrate to the separating gel with a basic pH, where the actual separation occurs. Stacking and separating different gels from different pore sizes (4-6Ã%,% T and 10-20Ã,% T), ionic strength and pH value (pH 6.8 or pH 8.8). The most commonly used electrolyte is the Tris-glycine-chloride buffer system containing SDS. At neutral pH, glycine primarily forms a zwitterionic form, at high pH glycines losing a positive charge and becoming anionic dominant. In the collection gel, smaller negatively charged chloride ions migrate in front of the protein (as the leading ions) and negative negatively charged, negatively charged, larger negatively charged ions migrate behind the protein (as the initial trailing ion), whereas in the gel separation ratio the second base of the ion migrates in front of the protein. The pH gradient between buffer gel stacking and separation causes the accumulation effect at the limit of the accumulating gel to the separation gel, since the glycinate partially loses its positive charge which slows down as pH increases and then, as previously left ion, follows the protein and becomes the leading ion, Different protein bands (seen after staining) become narrower and sharper - the effects of stacking. For the separation of smaller proteins and peptides, the TRIS-Tricine buffering system of SchÃÆ'¤gger and von Jagow is used because of higher protein spreads in the range of 0.5 to 50 KDa.
Gel coloring
At the end of the electrophoretic separation, all proteins are sorted by size and then can be analyzed by other methods, e. g. dyeing of proteins such as Coomassie staining (easy to use, most commonly used), silver staining (highest sensitivity), stain all staining, black amido stain 10B, fast green FCF staining, fluorescence stains such as epicokonon stains and orange dyeing SYPRO, and immunology detection such as Western Blot. The fluorescent dye has a relatively higher linearity between the protein quantity and the color intensity about threefold above the detection limit, i. e. the amount of protein can be estimated with the intensity of the color. When using tricloroethanol fluorescent protein dye, subsequent protein staining is removed when added to gel and gel solution irradiated with UV light after electrophoresis.
Analysis
The protein staining in the gel creates a documented banding pattern of various proteins. Glycoproteins have different levels of glycosylation and absorb the SDS more unevenly on glycosylation, resulting in wider and fuzzier bands. Membrane proteins, because their transmembrane domains, often consist of more hydrophobic amino acids, have lower solubility in aqueous solutions, tend to bind lipids, and tend to precipitate in aqueous solutions due to a hydrophobic effect when sufficient quantities of detergent do not exist.. This rainfall manifests itself to membrane proteins in SDS-PAGE in "tailings" above the transmembrane protein band. In this case, more SDS may be used (by using more or more concentrated sample buffers) and the amount of protein in the sample application can be reduced. Overloading gels with dissolved proteins produce these protein semicircular ribbons (for example in the image marker path at 66 KDa), allowing other proteins of the same molecular weight to be covered. The low contrast (as in the image marking line) between bands in the strip indicates either the presence of many proteins (low purity) or, if using purified and low-contrast proteins occurs only under one band, this indicates proteolytic degradation. of the protein, which first leads to band degradation, and after further degradation produces a homogeneous color ("smear") below the band. Bandbon pattern documentation is usually done by shooting or scanning. For subsequent recovery of molecules in individual bands, gel extraction may be performed.
Archiving
After protein staining and ribbon pattern documentation, the polyacrylamide gel may be dried for archival storage. Proteins can be extracted from them in the future. The gel is placed in a drying frame (with or without using heat) or in a vacuum dryer. The drying frame comprises two parts, one of which serves as the basis for wet cellophane films added by gel and one percent glycerol solution. Then a second wet selopant film is applied without bubbles, the second frame part is placed on top and the frame is sealed with a clip. Air bubble removal avoids gel fragmentation during drying. Water evaporates through a cellophane film. In contrast to the drying frame, the vacuum dryer produces a vacuum and heats the gel up to about 50 ° C.
Molecular mass determination
For a more accurate determination of the molecular weight, the relative migration distance of the individual protein bands is measured in the separating gel. Measurements are usually done in triplicate to improve accuracy. Relative mobility (called Rf value or Rm value) is the result of protein banding distance and distance from the front buffer. The distance of the tape and the front of each buffer is measured from the beginning of the separation gel. The buffer front spacing is approximately corresponding to the blue distance of bromophenol present in the sample buffer. The relative distance of the size plotting protein is plotted semi logarithmically against the known molecular weight. By comparison with the linear portion of the resulting graph or by regression analysis, the unknown molecular weight of the protein can be determined by its relative mobility. Band proteins with glycosylation may become opaque. Proteins with many basic amino acids (eg G. Histones) can cause overestimation of molecular weight or even not migrate to the gel at all, as they move more slowly in electrophoresis because of positive charges or even in the opposite direction. Thus, many acidic amino acids can cause rapid protein migration and underestimate the molecular mass.
Apps
SDS-PAGE combined with protein stains is widely used in biochemistry for rapid and precise separation and subsequent protein analysis. It has relatively low cost instruments and reagents and is an easy-to-use method. Because of its low scalability, it is widely used for analytical purposes and less for preparative purposes, especially when large numbers of proteins must be isolated.
In addition, SDS-PAGE is used in combination with a western blot for determining the presence of a particular protein in the protein mixture - or for post-translational modification analysis. Post-translational protein modification can cause different relative mobility (ie band shift ) or binding of the detection antibody used in the western blot (ie a missing or emerging band).
In protein mass spectrometry, SDS-PAGE is a widely used method for sample preparation before spectrometry, mostly using digestion in the gel. In terms of determining the mass of protein molecules, SDS-PAGE is slightly more precise than analytical ultracentrifugation, but less precise than mass spectrometry or - ignoring post-translational modification - a calculation of the mass of protein molecules from DNA. order.
In medical diagnostics, SDS-PAGE is used as part of the HIV test and to evaluate proteinuria. In HIV testing, HIV proteins are separated by SDS-PAGE and then detected by Western Blot with HIV-specific antibodies of patients, if they are present in their blood serum. SDS-PAGE for proteinuria evaluates the levels of various serum proteins in the urine, eg Albumin, Alpha-2-macroglobulin and IgG.
Variant
SDS-PAGE is the most widely used method for separation of protein electrophoretic gel. Two-dimensional gel electrophoresis sequentially combines the isoelectric focus or BAC-PAGE with SDS-PAGE. Native PAGE is used if the original protein folds should be maintained. For membrane protein separation, BAC-PAGE or CTAB-PAGE can be used as an alternative to SDS-PAGE. For greater electrophoretic separation of protein complexes, agarose gel electrophoresis can be used, eg SDD-AGE. Some enzymes can be detected through their enzyme activity with zimography.
Alternative
While being one of the more appropriate and inexpensive methods of protein separation and analysis, SDS-PAGE changes the properties of proteins. If non-denaturing conditions are required, proteins are separated by the original PAGE or chromatographic methods are different from the subsequent photometric quantification, for example affinity chromatography (or even purification of tandem affinity), size exclusion chromatography, ion exchange chromatography. Proteins can also be separated by sizes in tangential or ultrafiltration filtering. Single proteins may be isolated from the mixture by affinity chromatography or by a drop-down test. Some initial and cost-effective methods but coarse separation are usually based on a series of extractions and deposits using cosmotropic molecules, eg ammonium sulphate precipitation and polyethyleneglycol precipitation.
History
In 1948, Arne Tiselius was awarded the Nobel Prize in Chemistry for the discovery of the principle of electrophoresis as the migration of atoms and molecules charged and dissolved in an electric field. The use of solid matrix (originally paper disk) in zone electrophoresis increases separation. Electrophoresis was interrupted in 1964 by L. Ornstein and B. J. Davis made it possible to increase the separation by the cumulative effect. The use of cross-polyacrylamide hydrogels, in contrast to previously used paper discs or starch gels, provides higher gel stability and no microbial decomposition. The denaturation effect of SDS in continuous polyacrylamide gel and consequent increase in resolution was first described in 1965 by David F. Summers in the working group of James E. Darnell to separate polyovirus proteins. The current variant of SDS-PAGE was described in 1970 by Ulrich K. Laemmli and was originally used to characterize proteins in the T4 bacteriophage head.
References
External links
- OpenWetWare: protocol for BisTris SDS-PAGE
Source of the article : Wikipedia
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