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60-450: SDS-PAGE is an electrophoresis method that allows protein separation by mass. The medium (also referred to as ′matrix′) is a polyacrylamide-based discontinuous gel. The polyacrylamide-gel is typically sandwiched between two glass plates in a slab gel . Although tube gels (in glass cylinders) were used historically, they were rapidly made obsolete with the invention of the more convenient slab gels. In addition, SDS ( sodium dodecyl sulfate )

120-606: A mass spectrometry or - ignoring post-translational modifications - a calculation of the protein molecular mass from the DNA sequence . In medical diagnostics, SDS-PAGE is used as part of the HIV test and to evaluate proteinuria . In the HIV test, HIV proteins are separated by SDS-PAGE and subsequently detected by Western Blot with HIV-specific antibodies of the patient, if they are present in his blood serum . SDS-PAGE for proteinuria evaluates

180-444: A tangential flow filtration or an ultrafiltration . Single proteins can be isolated from a mixture by affinity chromatography or by a pull-down assay . Some historically early and cost effective but crude separation methods usually based upon a series of extractions and precipitations using kosmotropic molecules, for example the ammonium sulfate precipitation and the polyethyleneglycol precipitation. In 1948, Arne Tiselius

240-405: A concave meniscus, or internal cohesion pulls the liquid down to form a convex meniscus. This phenomenon is important in transpirational pull in plants. When a tube of a narrow bore, often called a capillary tube, is dipped into a liquid and the liquid wets the tube (with zero contact angle), the liquid surface inside the tube forms a concave meniscus, which is a virtually spherical surface having

300-437: A contact angle of zero and the surface tension can be obtained by measuring the mass of the menisci. This is typically done with a Wilhelmy plate . When reading a depth scale on the side of an instrument filled with liquid, such as a water level device , the meniscus must be taken into account in order to obtain an accurate measurement. Depth must be measured with the meniscus at eye level (to eliminate parallax error ) and at

360-527: A different relative mobility (i.e. a band shift ) or to a change in the binding of a detection antibody used in the western blot (i.e. a band disappears or appears). In mass spectrometry of proteins, SDS-PAGE is a widely used method for sample preparation prior to spectrometry, mostly using in-gel digestion . In regards to determining the molecular mass of a protein, the SDS-PAGE is a bit more exact than an analytical ultracentrifugation , but less exact than

420-449: A larger separation range of the molecular masses. Commercial gel systems (so-called pre-cast gels ) usually use the buffer substance Bis-tris methane with a pH value between 6.4 and 7.2 both in the stacking gel and in the separating gel. These gels are delivered cast and ready-to-use. Since they use only one buffer ( continuous gel electrophoresis ) and have a nearly neutral pH, they can be stored for several weeks. The more neutral pH slows

480-421: A later date. The gel is either placed in a drying frame (with or without the use of heat) or in a vacuum dryer. The drying frame consists of two parts, one of which serves as a base for a wet cellophane film to which the gel and a one percent glycerol solution are added. Then a second wet cellophane film is applied bubble-free, the second frame part is put on top and the frame is sealed with clips. The removal of

540-448: A migration of negatively charged molecules through the gel in the direction of the positively charged anode . The gel acts like a sieve. Small proteins migrate relatively easily through the mesh of the gel, while larger proteins are more likely to be retained and thereby migrate more slowly through the gel, thereby allowing proteins to be separated by molecular size. The electrophoresis lasts between half an hour to several hours depending on

600-402: A mixing of the buffers. The gel is produced by free radical polymerization in a mold consisting of two sealed glass plates with spacers between the glass plates. In a typical mini-gel setting, the spacers have a thickness of 0.75 mm or 1.5 mm, which determines the loading capacity of the gel. For pouring the gel solution, the plates are usually clamped in a stand which temporarily seals

660-445: A separating gel. Upon application of a constant electric field, the proteins migrate towards the anode, each with a different speed, depending on their mass. This simple procedure allows precise protein separation by mass. SDS tends to form spherical micelles in aqueous solutions above a certain concentration called the critical micellar concentration (CMC). Above the critical micellar concentration of 7 to 10 millimolar in solutions,

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720-432: Is an SDS-containing Tris - glycine - chloride buffer system. At neutral pH, glycine predominantly forms the zwitterionic form, at high pH the glycines lose positive charges and become predominantly anionic. In the collection gel, the smaller, negatively charged chloride ions migrate in front of the proteins (as leading ions) and the slightly larger, negatively and partially positively charged glycinate ions migrate behind

780-475: Is an easy-to-use method. Because of its low scalability , it is mostly used for analytical purposes and less for preparative purposes, especially when larger amounts of a protein are to be isolated. Additionally, SDS-PAGE is used in combination with the western blot for the determination of the presence of a specific protein in a mixture of proteins - or for the analysis of post-translational modifications . Post-translational modifications of proteins can lead to

840-401: Is carefully pulled out after polymerisation, leaving pockets for the sample application. For later use of proteins for protein sequencing , the gels are often prepared the day before electrophoresis to reduce reactions of unpolymerised acrylamide with cysteines in proteins. By using a gradient mixer , gradient gels with a gradient of acrylamide (usually from 4 to 12%) can be cast, which have

900-416: Is defined as the distance migrated by the protein band divided by the distance migrated by the buffer front. The distances are each measured from the beginning of the separation gel. The migration of the buffer front roughly corresponds to the migration of the dye contained in the sample buffer. The Rf's of the size marker are plotted semi-logarithmically against their known molecular weights. By comparison with

960-552: Is significantly different from the classic particle electrophoresis because of droplet characteristics such as a mobile surface charge and the nonrigidity of the interface. Also, the liquid–liquid system, where there is an interplay between the hydrodynamic and electrokinetic forces in both phases, adds to the complexity of electrophoretic motion. Suspended particles have an electric surface charge , strongly affected by surface adsorbed species, on which an external electric field exerts an electrostatic Coulomb force . According to

1020-409: Is sometimes called anaphoresis . Electrophoresis is the basis for analytical techniques used in biochemistry for separating particles, molecules, or ions by size , charge, or binding affinity either freely or through a supportive medium using a one-directional flow of electrical charge. It is used extensively in DNA , RNA and protein analysis. Liquid droplet electrophoresis

1080-444: Is started. The solution is then poured between the glass plates without creating bubbles. Depending on the amount of catalyst and radical starter and depending on the temperature, the polymerisation lasts between a quarter of an hour and several hours. The lower gel (separating gel) is poured first and covered with a few drops of a barely water-soluble alcohol (usually buffer-saturated butanol or isopropanol), which eliminates bubbles from

1140-409: Is used in laboratories to separate macromolecules based on their charges. The technique normally applies a negative charge called cathode so protein molecules move towards a positive charge called anode . Therefore, electrophoresis of positively charged particles or molecules ( cations ) is sometimes called cataphoresis , while electrophoresis of negatively charged particles or molecules (anions)

1200-457: Is used. About 1.4 grams of SDS bind to a gram of protein, corresponding to one SDS molecule charges per two amino acids . SDS acts as a surfactant , masking the protein's intrinsic charge and conferring them very similar charge-to-mass ratios. The intrinsic charges of the proteins are negligible in comparison to the SDS loading, and the positive charges are also greatly reduced in the basic pH range of

1260-471: Is valid for most aqueous systems, where the Debye length is usually only a few nanometers . It only breaks for nano-colloids in solution with ionic strength close to water. The Smoluchowski theory also neglects the contributions from surface conductivity . This is expressed in modern theory as condition of small Dukhin number : In the effort of expanding the range of validity of electrophoretic theories,

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1320-409: Is very powerful because it works for dispersed particles of any shape at any concentration . It has limitations on its validity. For instance, it does not include Debye length κ (units m). However, Debye length must be important for electrophoresis, as follows immediately from Figure 2, "Illustration of electrophoresis retardation" . Increasing thickness of the double layer (DL) leads to removing

1380-400: Is zero: Considering the drag on the moving particles due to the viscosity of the dispersant, in the case of low Reynolds number and moderate electric field strength E , the drift velocity of a dispersed particle v is simply proportional to the applied field, which leaves the electrophoretic mobility μ e defined as: The most well known and widely used theory of electrophoresis

1440-629: The SDD-AGE . Some enzymes can be detected via their enzyme activity by zymography . While being one of the more precise and low-cost protein separation and analysis methods, the SDS-PAGE denatures proteins. Where non-denaturing conditions are necessary, proteins are separated by a native PAGE or different chromatographic methods with subsequent photometric quantification , for example affinity chromatography (or even tandem affinity purification ), size exclusion chromatography , ion exchange chromatography . Proteins can also be separated by size in

1500-462: The Western Blot . The fluorescent dyes have a comparatively higher linearity between protein quantity and color intensity of about three orders of magnitude above the detection limit (the quantity of protein that can be estimated by color intensity). When using the fluorescent protein dye trichloroethanol , a subsequent protein staining is omitted if it was added to the gel solution and the gel

1560-405: The double layer theory, all surface charges in fluids are screened by a diffuse layer of ions, which has the same absolute charge but opposite sign with respect to that of the surface charge. The electric field also exerts a force on the ions in the diffuse layer which has direction opposite to that acting on the surface charge . This latter force is not actually applied to the particle, but to

1620-417: The ions in the diffuse layer located at some distance from the particle surface, and part of it is transferred all the way to the particle surface through viscous stress . This part of the force is also called electrophoretic retardation force, or ERF in short. When the electric field is applied and the charged particle to be analyzed is at steady movement through the diffuse layer, the total resulting force

1680-434: The meniscus and protects the gel solution of the radical scavenger oxygen. After the polymerisation of the separating gel, the alcohol is discarded and the residual alcohol is removed with filter paper . After addition of APS and TEMED to the stacking gel solution, it is poured on top of the solid separation gel. Afterwards, a suitable sample comb is inserted between the glass plates without creating bubbles. The sample comb

1740-595: The SDS simultaneously occurs as single molecules ( monomer ) and as micelles, below the CMC SDS occurs only as monomers in aqueous solutions. At the critical micellar concentration, a micelle consists of about 62 SDS molecules. However, only SDS monomers bind to proteins via hydrophobic interactions, whereas the SDS micelles are anionic on the outside and do not adsorb any protein. SDS is amphipathic in nature, which allows it to unfold both polar and nonpolar sections of protein structure. In SDS concentrations above 0.1 millimolar,

1800-409: The SDS-PAGE was described in 1970 by Ulrich K. Laemmli and initially used to characterise the proteins in the head of bacteriophage T4 . Electrophoresis Electrophoresis is the motion of charged dispersed particles or dissolved charged molecules relative to a fluid under the influence of a spatially uniform electric field . As a rule, these are zwitterions . Electrophoresis

1860-403: The adhesion energy is less than half the cohesion energy. Convex menisci occur, for example, between mercury and glass in barometers and thermometers . In general, the shape of the surface of a liquid can be complex. For a sufficiently narrow tube with circular cross-section, the shape of the meniscus will approximate a section of a spherical surface , while for a large container, most of

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1920-511: The air bubbles avoids a fragmentation of the gel during drying. The water evaporates through the cellophane film. In contrast to the drying frame, a vacuum dryer generates a vacuum and heats the gel to about 50 °C. For a more accurate determination of the molecular weight, the relative migration distances of the individual protein bands are measured in the separating gel. The measurements are usually performed in triplicate for increased accuracy. The relative mobility (called Rf value or Rm value)

1980-429: The cationic surfactants CTAB in a CTAB-PAGE, or 16-BAC in a BAC-PAGE. The SDS-PAGE method is composed of gel preparation, sample preparation, electrophoresis, protein staining or western blotting and analysis of the generated banding pattern. When using different buffers in the gel (discontinuous gel electrophoresis), the gels are made up to one day prior to electrophoresis, so that the diffusion does not lead to

2040-401: The center of the meniscus, i.e. the top of a convex meniscus or the bottom of a concave meniscus. Manufacturers of glassware and other tools calibrate their measurement marks to account for the meniscus. This means that any instrument is calibrated for a specific liquid, usually water. Menisci are a manifestation of capillary action , by which either surface adhesion pulls a liquid up to form

2100-409: The container ( adhesion ) is more than half the attraction of the particles of the liquid to each other ( cohesion ), causing the liquid to climb the walls of the container (see Surface tension § Causes ). This occurs between water and glass. Water-based fluids like sap, honey, and milk also have a concave meniscus in glass or other wettable containers. Conversely, a convex meniscus occurs when

2160-617: The electric field must be modeled spatially, tracking its magnitude and direction. Poisson's equation can be used to model this spatially-varying electric field. Its influence on fluid flow can be modeled with the Stokes law , while transport of different ions can be modeled using the Nernst–Planck equation . This combined approach is referred to as the Poisson-Nernst-Planck-Stokes equations. It has been validated for

2220-430: The electrophoresis due to the positive charges or even to the opposite direction. On the other hand, many acidic amino acids can lead to accelerated migration of a protein and an underestimation of its molecular mass. The SDS-PAGE in combination with a protein stain is widely used in biochemistry for the quick and exact separation and subsequent analysis of proteins. It has comparatively low instrument and reagent costs and

2280-402: The electrophoresis of particles. Meniscus (liquid) In physics (particularly fluid statics ), the meniscus ( pl. : menisci , from Greek  'crescent') is the curve in the upper surface of a liquid close to the surface of the container or another object, produced by surface tension . A concave meniscus occurs when the attraction between the particles of the liquid and

2340-423: The gel creates a documentable banding pattern of the various proteins. The documentation of the banding pattern is usually done by photographing or scanning. For a subsequent recovery of the molecules in individual bands, a gel extraction can be performed. After protein staining and documentation of the banding pattern, the polyacrylamide gel can be dried for archival storage. Proteins can be extracted from it at

2400-458: The hydrolysis and thus the decomposition of the polyacrylamide. Furthermore, there are fewer acrylamide-modified cysteines in the proteins. Due to the constant pH in collecting and separating gel there is no stacking effect. Proteins in BisTris gels can not be stained with ruthenium complexes. This gel system has a comparatively large separation range, which can be varied by using MES or MOPS in

2460-584: The levels of various serum proteins in the urine, e.g. Albumin , Alpha-2-macroglobulin and IgG . SDS-PAGE is the most widely used method for gel electrophoretic separation of proteins. Two-dimensional gel electrophoresis sequentially combines isoelectric focusing or BAC-PAGE with a SDS-PAGE. Native PAGE is used if native protein folding is to be maintained. For separation of membrane proteins, BAC-PAGE or CTAB-PAGE may be used as an alternative to SDS-PAGE. For electrophoretic separation of larger protein complexes, agarose gel electrophoresis can be used, e.g.

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2520-429: The linear part of the generated graph or by a regression analysis, the molecular weight of an unknown protein can be determined by its relative mobility. Bands of proteins with glycosylations can be blurred, as glycosylation is often heterogenous. Proteins with many basic amino acids (e. g. histones ) can lead to an overestimation of the molecular weight or even not migrate into the gel at all, because they move slower in

2580-618: The migrating colored band, the electrophoresis can be stopped before the dye and also the samples have completely migrated through the gel and leave it. The most commonly used method is the discontinuous SDS-PAGE. In this method, the proteins migrate first into a collecting gel with neutral pH, in which they are concentrated and then they migrate into a separating gel with basic pH, in which the actual separation takes place. Stacking and separating gels differ by different pore size (4-6 % T and 10-20 % T), ionic strength and pH values (pH 6.8 or pH 8.8). The electrolyte most frequently used

2640-450: The opposite asymptotic case was considered, when Debye length is larger than particle radius: Under this condition of a "thick double layer", Erich Hückel predicted the following relation for electrophoretic mobility: This model can be useful for some nanoparticles and non-polar fluids, where Debye length is much larger than in the usual cases. There are several analytical theories that incorporate surface conductivity and eliminate

2700-399: The otherwise open underside of the glass plates with the two spacers. For the gel solution, acrylamide is mixed as gel-former (usually 4% V/V in the stacking gel and 10-12 % in the separating gel), methylenebisacrylamide as a cross-linker, stacking or separating gel buffer, water and SDS. By adding the catalyst TEMED and the radical initiator ammonium persulfate (APS) the polymerisation

2760-570: The point of retardation force further from the particle surface. The thicker the DL, the smaller the retardation force must be. Detailed theoretical analysis proved that the Smoluchowski theory is valid only for sufficiently thin DL, when particle radius a is much greater than the Debye length: This model of "thin double layer" offers tremendous simplifications not only for electrophoresis theory but for many other electrokinetic theories. This model

2820-729: The presence of SDS (the latter, however, only at room temperature). To denature the SDS-resistant complexes a high activation energy is required, which is achieved by heating. SDS resistance is based on a metastability of the protein fold. Although the native, fully folded, SDS-resistant protein does not have sufficient stability in the presence of SDS, the chemical equilibrium of denaturation at room temperature occurs slowly. Stable protein complexes are characterised not only by SDS resistance but also by stability against proteases and an increased biological half-life . Alternatively, polyacrylamide gel electrophoresis can also be performed with

2880-415: The proteins (as initial trailing ions), whereas in the comparatively basic separating gel both ions migrate in front of the proteins. The pH gradient between the stacking and separation gel buffers leads to a stacking effect at the border of the stacking gel to the separation gel, since the glycinate partially loses its slowing positive charges as the pH increases and then, as the former trailing ion, overtakes

2940-857: The proteins and becomes a leading ion, which causes the bands of the different proteins (visible after a staining) to become narrower and sharper - the stacking effect. For the separation of smaller proteins and peptides, the TRIS- Tricine buffer system of Schägger and von Jagow is used due to the higher spread of the proteins in the range of 0.5 to 50 kDa. At the end of the electrophoretic separation, all proteins are sorted by size and can then be analyzed by other methods, e. g. protein staining such as Coomassie staining (most common and easy to use), silver staining (highest sensitivity), stains all staining, Amido black 10B staining, Fast green FCF staining, fluorescent stains such as epicocconone stain and SYPRO orange stain, and immunological detection such as

3000-420: The proteins in the actual samples, which migrate in parallel in different tracks of the gel. The size marker is often pipetted into the first or last pocket of a gel. For separation, the denatured samples are loaded onto a gel of polyacrylamide, which is placed in an electrophoresis buffer with suitable electrolytes. Thereafter, a voltage (usually around 100 V, 10-20 V per cm gel length) is applied, which causes

3060-441: The restriction of a small Dukhin number, pioneered by Theodoor Overbeek and F. Booth. Modern, rigorous theories valid for any Zeta potential and often any aκ stem mostly from Dukhin–Semenikhin theory. In the thin double layer limit, these theories confirm the numerical solution to the problem provided by Richard W. O'Brien and Lee R. White. For modeling more complex scenarios, these simplifications become inaccurate, and

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3120-658: The running buffer. During sample preparation, the sample buffer, and thus SDS, is added in excess to the proteins, and the sample is then heated to 95 °C for five minutes, or alternatively 70 °C for ten minutes. Heating disrupts the secondary and tertiary structures of the protein by disrupting hydrogen bonds and stretching the molecules. Optionally, disulfide bridges can be cleaved by reduction. For this purpose, reducing thiols such as β-mercaptoethanol (β-ME, 5% by volume), dithiothreitol (DTT, 10–100 millimolar), dithioerythritol (DTE, 10 millimolar), tris(2-carboxyethyl)phosphine or tributylphosphine are added to

3180-408: The sample buffer. After cooling to room temperature, each sample is pipetted into its own well in the gel, which was previously immersed in electrophoresis buffer in the electrophoresis apparatus. In addition to the samples, a molecular-weight size marker is usually loaded onto the gel. This consists of proteins of known sizes and thereby allows the estimation (with an error of ± 10%) of the sizes of

3240-461: The stacking effect. The use of cross-linked polyacrylamide hydrogels, in contrast to the previously used paper discs or starch gels, provided a higher stability of the gel and no microbial decomposition. The denaturing effect of SDS in continuous polyacrylamide gels and the consequent improvement in resolution was first described in 1965 by David F. Summers in the working group of James E. Darnell to separate poliovirus proteins. The current variant of

3300-401: The unfolding of proteins begins, and above 1 mM, most proteins are denatured. Due to the strong denaturing effect of SDS and the subsequent dissociation of protein complexes, quaternary structures can generally not be determined with SDS. Exceptions are proteins that are stabilised by covalent cross-linking (e.g. -S-S- linkages) and the SDS-resistant protein complexes, which are stable even in

3360-411: The upper surface of the liquid will be almost flat, only curving up (if concave) or down (if convex) near the edges. The formation of menisci is commonly used in surface science to measure contact angles and surface tension . In a contact angle measurement, the shape of the menisci is measured with a balance or optically with a digital camera. In a surface tension measurement, the measurement probe has

3420-491: The voltage and length of gel used. The fastest-migrating proteins (with a molecular weight of less than 5 kDa) form the buffer front together with the anionic components of the electrophoresis buffer, which also migrate through the gel. The area of the buffer front is made visible by adding the comparatively small, anionic dye bromophenol blue to the sample buffer. Due to the relatively small molecule size of bromophenol blue, it migrates faster than proteins. By optical control of

3480-517: Was awarded the Nobel Prize in Chemistry for the discovery of the principle of electrophoresis as the migration of charged and dissolved atoms or molecules in an electric field. The use of a solid matrix (initially paper discs) in a zone electrophoresis improved the separation. The discontinuous electrophoresis of 1964 by L. Ornstein and B. J. Davis made it possible to improve the separation by

3540-401: Was developed in 1903 by Marian Smoluchowski : where ε r is the dielectric constant of the dispersion medium , ε 0 is the permittivity of free space (C  N  m ), η is dynamic viscosity of the dispersion medium (Pa s), and ζ is zeta potential (i.e., the electrokinetic potential of the slipping plane in the double layer , units mV or V). The Smoluchowski theory

3600-457: Was irradiated with UV light after electrophoresis. In Coomassie staining, gel is fixed in a 50% ethanol 10% glacial acetic acid solution for 1 hr. Then the solution is changed for fresh one and after 1 to 12 hrs gel is changed to a staining solution (50% methanol, 10% glacial acetic acid, 0.1% coomassie brilliant blue) followed by destaining changing several times a destaining solution of 40% methanol, 10% glacial acetic acid. Protein staining in

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