Polyacrylamide gel electrophoresis
SDS -PAGE ( sodium dodecyl sulfate Abbreviation for polyacrylamide gel electrophoresis, sodium dodecyl sulfate - polyacrylamide gel electrophoresis) is a variant of polyacrylamide gel electrophoresis, an analytical method biochemistry for separation of mixtures by molecular mass in an electric field.
Area of application
SDS-PAGE is used to analyze proteins. A separation medium (also referred to as a matrix ) is used for this type of electrophoresis, a discontinuous polyacrylamide gel. In addition, SDS ( sodium dodecyl sulfate) is used. This anionic surfactant ( detergent) covers the intrinsic charge of proteins. Per gram of protein bind approximately 1.4 grams of SDS, so that the proteins have a constant negative charge distribution. The negative charges of SDS lead to mutual repulsion, which together with the denaturation by boiling to a linearization of the proteins. This allows the separation according to the chain length, proportional to the molecular weight, since the longer the gel proteins are retained more strongly than shorter proteins. The SDS surrounds the linearized protein with an approximately ellipsoidal shell. The natural charges of the proteins are negligible under the SDS- loading, the positive charges are also in the basic pH of the separating gel according to Laemmli greatly reduced.
Method
SDS during sample preparation is added in excess of the proteins, and the sample was then heated to 95 ° C to break the secondary and tertiary structures by interrupting hydrogen bonding, and the stretching of the molecules. Optional disulfide bonds can be cleaved by reduction. To reducing thiols such as β -mercaptoethanol, dithiothreitol ( DTT) or dithioerythritol to (DTE ) were added to the sample buffer. At the end of this preparation loaded with SDS proteins have an ellipsoidal shape. Because of the strong Denaturierungseffekts no quaternary structures can be generally determined with SDS. Exceptions to this are, for example, previously stabilized by covalent cross-linking proteins and the so-called SDS-resistant protein complexes, (the latter only at room temperature) which are stable even in the presence of SDS. The SDS- resistance based on the metastability. To denature the resistant complexes, a high activation energy is needed. Although the native, correctly folded protein does not have sufficient stability under the conditions that the chemical equilibrium of the denaturation is a slow. Stable protein complexes are distinguished not only from the SDS- resistance also by stability against proteases and increased biological half-life.
For separating the denatured samples are loaded onto a polyacrylamide gel, which is inserted in suitable electrolytes. Then an electrical voltage is applied which causes a migration of the negatively charged specimen through the gel. The gel acts like a sieve. Small proteins migrate relatively easily through the mesh of the gel, whereas large proteins tend to be retained and thus migrate more slowly through the gel. At the end of the process, all proteins are sorted by size and can proceed ( dyes such as Coomassie staining, silver staining, the Fast Green FCF staining, SYPRO Orange staining and immunological tests such as the Western blot) are made visible. In addition to the samples usually a size marker is loaded on the gel. This consists of proteins of known size, thus enabling the estimation of the size of the proteins in the actual samples.
The method most commonly used is the discontinuous SDS-PAGE. In this, the proteins first hike in a stacking gel with a neutral pH, where they are concentrated and then treated with alkaline pH, in which the actual separation takes place in a separating gel. Collection and separation gel differ in their pore size and pH values . The pH gradient between collection and resolving gel buffer leads to a stacking effect on the border separating gel. As an electrolyte a SDS -containing Tris-glycine buffer system is often used. Developed by Ulrich Laemmli discontinuous system enables a good separation of proteins with molecular masses between 5 and 250 KDa. The publication, which has been described, is the most widely cited paper by an individual author, and the second most cited overall. For the separation of small proteins and peptides is due to the greater spread of the proteins in the range from 1 to 50 KDa, the TRIS - Tricine buffer system of Schagger and von Jagow.
Glycoproteins adsorb SDS to the glycosylations uneven, resulting in broader and less sharp bands. Membrane proteins are constructed because of their transmembrane domain often from hydrophobic amino acids, have a lower solubility, tend to bind due to hydrophobic effects lipids and tend to precipitate in aqueous solutions if insufficient detergent is present. This precipitation is expressed in membrane proteins in SDS -PAGE in a " tailing " above the band of the transmembrane protein, whereas more SDS can be used ( by using more or more concentrated sample buffer ) and the amount of protein are reduced in the gel application. Overloading the gel with a soluble protein expressed in a semi-circular band of this protein (eg, in the Marker track the figure at 66 KDa ). A low contrast (like in the Marker track the figure) between gangs within a lane indicates either the presence of many proteins with intermediate molecular weights down ( low purity ) or if, in purified proteins, a lack of contrast occurs only below a band, a proteolytic degradation through the protein, which is expressed first in dismantling gangs and after further degradation in a homogeneous staining ( "smear" ) below a gang.
Commercial gel systems
Commercial gel systems ( so-called pre -cast gels) mostly use the buffer substance BisTris with a pH of 6.4 in both the collection and in the separating gel. These gels are supplied already poured finished and ready for use immediately. The lower pH prevents the disintegration of the polyacrylamide gels and makes for a long time shelf life. Additionally, this gel system has a very large separation range, can also be varied by the use of MES or MOPS in the running buffer.
Molar Mass
For a more accurate determination of molar mass, the relative run lengths of the individual protein bands are measured in the gel. The relative amount of tracking ( also Rf, Rm value ) is the quotient of the run length in the considered protein and the run length of the protein front. The tracking of the protein front approximates the walking distance of the bromophenol blue in the sample buffer contained. The relative spacings in the proteins of the size marker are semilogarithmic plotted against their known molecular weights. About compared with the generated graph or calculated by a regression analysis, the molecular weight of an unknown protein can be determined by its relative spacing. Bands of proteins with glycosylation may be blurred. Proteins with many basic amino acids (eg histones) can lead to an overestimation of the molecular weight.