SDS-PAGE,
sodium dodecyl sulfateSodium 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...
polyacrylamidePolyacrylamide 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...
gel electrophoresisGel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
, describes a collection of related techniques widely used in
biochemistryBiochemistry, 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...
,
forensicsForensic 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...
,
geneticsGenetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
and
molecular biologyMolecular 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
proteinProteins 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 according to their
electrophoretic mobilityElectrophoresis, 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...
(a function of the length of a polypeptide chain and its charge). In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis.
Sample preparation
Samples may be any material containing proteins, such as for example prokaryotic or eukaryotic cells, tissues, viruses, environmental samples, or purified proteins. In the case of solid tissues, these are often first broken down mechanically using a
blenderA blender is a kitchen appliance for chopping or liquefying food.Blender may also refer to:Media:* Blender , a music-themed magazine* Blender , a free and open-source software program for 3D modeling, animation, and rendering...
(for larger sample volumes), using a
homogenizerA homogenizer is a piece of laboratory equipment used for the homogenization of various types of material, such as tissue, plant, food, soil, and many others. Many different models have been developed using various physical technologies for disruption. The 'mortar and pestle', already used for...
(smaller volumes), by sonicator or by using cycling of high pressure. Cells may also be broken open by one of the above mechanical methods.
In the case of tissues or cells, a combination of biochemical and mechanical techniques – including various types of filtration and
centrifugationA centrifuge is a piece of equipment, generally driven by an electric motor , that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis...
– may be used to separate different cell compartments and
organelleIn cell biology, an organelle is a specialized subunit within a cell that has a specific function, and is usually separately enclosed within its own lipid bilayer....
s prior to electrophoresis.
The sample to be analyzed is mixed with
SDSSodium 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...
, an anionic
detergentA 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...
which
denaturesDenaturation 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...
secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. Heating the samples to at least 60°C further promotes protein denaturation, helping SDS to bind.
A tracking dye may be added to the protein solution. This typically has a higher electrophoretic mobility than the proteins to allow the experimenter to track the progress of the protein solution through the gel during the electrophoretic run.
Preparing acrylamide gels
The gels typically consist of
acrylamideAcrylamide 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...
, bisacrylamide,
SDSSodium 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...
, and a buffer with an adjusted pH. The solution may be degassed under a vacuum to prevent the formation of air bubbles during polymerization.
A source of free radicals and a stabilizer such as ammonium persulfate and TEMED are added to initiate polymerization. The polymerization reaction results in a gel because of the added bisacrylamide, generally about 1 part in 35 relative to acrylamide, which can form cross-links between two polyacrylamide molecules. The ratio of acrylamide to bisacrylamide can be varied for special purposes. The acrylamide concentration of the gel can also be varied, generally in the range from 5% to 25%. Lower percentage gels are better for resolving very high molecular weight proteins, while much higher percentages are needed to resolve smaller proteins. Determining how much of the various solutions to mix together to make gels of particular acrylamide concentration is possible with this
on line calculator from
EnCor Biotechnology Inc.EnCor Biotechnology Incorporated is a company founded in Gainesville, Florida as a spin-off from the University of Florida. It arose as a result of the work of Gerry Shaw, a British scientist and professor in the University. Dr...
.
Gels are usually polymerized between two glass plates in a gel caster, with a comb inserted at the top to create the sample wells. After the gel is polymerized the comb can be removed and the gel is ready for electrophoresis.
Electrophoresis
Various buffer systems are used in SDS-PAGE depending on the nature of the sample and the experimental objective. The buffers used at the anode and cathode may be the same or different.
An electric field is applied across the gel, causing the negatively-charged proteins to migrate across the gel towards the positive (+) electrode (anode). Depending on their size, each protein will move differently through the gel matrix: short proteins will more easily fit through the
poresWiktionary is a multilingual, web-based project to create a free content dictionary, available in 158 languages...
in the gel, while larger ones will have more difficulty (they encounter more resistance). After a set amount of time (usually a few hours- though this depends on the voltage applied across the gel; higher voltages run faster but tend to produce somewhat poorer resolution), the proteins will have differentially migrated based on their size; smaller proteins will have traveled farther down the gel, while larger ones will have remained closer to the point of origin. Proteins may therefore be separated roughly according to size (and thus molecular weight); however certain
glycoproteinGlycoproteins are proteins that contain oligosaccharide chains covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending...
s behave anomalously on SDS gels.
Further processing
Following electrophoresis, the gel may be stained (most commonly with Coomassie Brilliant Blue R-250 or silver stain), allowing visualization of the separated proteins, or processed further (e.g.
Western blotThe 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...
). After staining, different proteins will appear as distinct bands within the gel. It is common to run
molecular weight size markerA 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 of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the approximate
molecular massThe molecular mass of a substance is the mass of one molecule of that substance, in unified atomic mass unit u...
of unknown proteins by comparing the distance travelled relative to the marker.
SDS-PAGE is usually the first choice as an assay of protein purity due to its reliability and ease. The presence of SDS and the denaturing step causes proteins to be separated approximately based on size, but aberrant migration of some proteins may occur. Different proteins may also stain differently, which interferes with quantification by staining. SDS-PAGE may also be used as a preparative technique for the purification of proteins. For example,
quantitative
preparative
native
continuous
poly
acrylamide
gel
electrophoresis (
QPNC-PAGEQPNC-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...
) is a method for separating native
metalloproteinMetalloprotein is a generic term for a protein that contains a metal ion cofactor. Metalloproteins have many different functions in cells, such as enzymes, transport and storage proteins, and signal transduction proteins. Indeed, about one quarter to one third of all proteins require metals to...
s in complex biological matrices.
Chemical ingredients and their roles
Polyacrylamide gel (PAG) had been known as a potential embedding medium for sectioning tissues as early as 1964, and two independent groups employed PAG in electrophoresis in 1959. It possesses several electrophoretically desirable features that make it a versatile medium. It is a synthetic, thermo-stable, transparent, strong, chemically relatively inert gel, and can be prepared with a wide range of average pore sizes. The pore size of a gel is determined by two factors, the total amount of acrylamide present (%T) (T = Total concentration of acrylamide and bisacrylamide monomer) and the amount of cross-linker (%C) (C = bisacrylamide concentration). Pore size decreases with increasing %T; with cross-linking, 5%C gives the smallest pore size. Any increase or decrease in %C from 5% increases the pore size, as pore size with respect to %C is a parabolic function with vertex as 5%C. This appears to be because of non-homogeneous bundling of polymer strands within the gel. This gel material can also withstand high
voltageVoltage, otherwise known as electrical potential difference or electric tension is the difference in electric potential between two points — or the difference in electric potential energy per unit charge between two points...
gradients, is amenable to various staining and destaining procedures, and can be digested to extract separated fractions or dried for autoradiography and permanent recording.
Components
- Chemical buffer
Buffer may refer to:*Buffer state, a country lying between two potentially hostile greater powers, thought to prevent conflict between them* Buffer zone, any area that keeps two or more other areas distant from one another, may be demilitarized...
Stabilizes the pH value to the desired value within the gel itself and in the electrophoresis buffer. The choice of buffer also affects the electrophoretic mobility of the buffer counterionA counterion is the ion that accompanies an ionic species in order to maintain electric neutrality. In table salt the sodium cation is the counterion for the chlorine anion and vice versa.In a charged transition metal complex, a simple A counterion is the ion that accompanies an ionic species in...
s and thereby the resolution of the gel. The buffer should also be unreactive and not modify of react with most proteins. Different buffers may be used as cathode and anode buffers, respectively, depending on the application. Multiple pH values may be used within a single gel, for example in DISC electrophoresis. Common buffers in SDS-PAGE include TrisTris is an abbreviation of the organic compound known as trisaminomethane, with the formula 3CNH2. Tris is extensively used in biochemistry and molecular biology. In biochemistry, tris is widely used as a component of buffer solutions, such as in TAE and TBE buffer, especially for solutions of...
, Bis-Tris, or imidazoleImidazole is an organic compound with the formula C3H4N2. This aromatic heterocyclic is a diazole and is classified as an alkaloid. Imidazole refers to the parent compound, whereas imidazoles are a class of heterocycles with similar ring structure, but varying substituents...
.
- Counterion
A counterion is the ion that accompanies an ionic species in order to maintain electric neutrality. In table salt the sodium cation is the counterion for the chlorine anion and vice versa.In a charged transition metal complex, a simple A counterion is the ion that accompanies an ionic species in...
balance the intrinsic charge of the buffer ion and also affect the electric field strength during electrophoresis. Highly charged and mobile ions are often avoided in SDS-PAGE cathode buffers, but may be included in the gel itself, where it migrates ahead of the protein. In applications such as DISC SDS-PAGE the pH values within the gel may vary to change the average charge of the counterions during the run to improve resolution. Popular counterions are glycineGlycine is an organic compound with the formula NH2CH2COOH. Having a hydrogen substituent as its 'side chain', glycine is the smallest of the 20 amino acids commonly found in proteins. Its codons are GGU, GGC, GGA, GGG cf. the genetic code.Glycine is a colourless, sweet-tasting crystalline solid...
and tricineTricine is an organic compound that is used in buffer solutions. The name tricine comes from tris and glycine from which it was derived. It is a zwitterionic amino acid with a useful buffering range of pH 7.4-8.8. It is a white crystalline powder that is moderately soluble in water. It has a pH...
. Glycine has been used as the source of trailing ion or slow ion because its pKa is 9.69 and mobility of glycinate are such that the effective mobility can be set at a value below that of the slowest known proteins of net negative chargeElectric charge is a physical property of matter that causes it to experience a force when near other electrically charged matter. Electric charge comes in two types, called positive and negative. Two positively charged substances, or objects, experience a mutual repulsive force, as do two...
in the pH range. The minimum pH of this range is approximately 8.0.[
- 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...
(C3H5NO; mW: 71.08). When dissolved in water, slow, spontaneous autopolymerizationIn polymer chemistry, polymerization is a process of reacting monomer molecules together in a chemical reaction to form three-dimensional networks or polymer chains...
of acrylamide takes place,joining molecules together by head on tail fashion to form long single-chain polymers. The presence of a free radical-generating system greatly accelerates polymerization. This kind of reaction is known as VinylA vinyl compound is any organic compound that contains a vinyl group ,which are derivatives of ethene, CH2=CH2, with one hydrogen atom replaced with some other group...
addition polymerisationChain growth polymerization is a polymerization technique where unsaturated monomer molecules add on to a growing polymer chain one at a time...
. A solution of these polymer chains becomes viscous but does not form a gel, because the chains simply slide over one another. Gel formation requires linking various chains together. Acrylamide is a neurotoxinA 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...
. It is also essential to store acrylamide in a cool dark and dry place to reduce autopolymerisation and hydrolysisHydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
.
- Bisacrylamide (N,N'-Methylenebisacrylamide) (C7H10N2O2; mW: 154.17). Bisacrylamide is the most frequently used cross linking agent for polyacrylamide gels. Chemically it can be thought of as two acrylamide molecules coupled head to head at their non-reactive ends. Bisacrylamide can crosslink two polyacrylamide chains to one another, thereby resulting in a gel.
- 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...
(SDS) (C12H25NaO4S; mW: 288.38). SDS is a strong detergent agent used to denature native proteins to unfolded, individual polypeptides. When a protein mixture is heated to 100 °C in presence of SDS, the detergentA 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...
wraps around the polypeptide backbone. It binds to polypeptides in a constant weight ratio of 1.4 g SDS/g of polypeptide. In this process, the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS. Thus polypeptides after treatment become rod-like structures possessing a uniform charge density, that is same net negative charge per unit length. The electrophoretic mobilities of these proteins will be a linear function of the logarithmThe logarithm of a number is the exponent by which another fixed value, the base, has to be raised to produce that number. For example, the logarithm of 1000 to base 10 is 3, because 1000 is 10 to the power 3: More generally, if x = by, then y is the logarithm of x to base b, and is written...
s of their molecular weights.
- Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass-charge ratio, as each protein has an isoelectric point
The isoelectric point , sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge....
and molecular weight particular to its primary structureThe primary structure of peptides and proteins refers to the linear sequence of its amino acid structural units. The term "primary structure" was first coined by Linderstrøm-Lang in 1951...
. This is known as Native PAGE. Adding SDS solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide.
- Ammonium persulfate
Ammonium persulfate 2S2O8 is a strong oxidizing agent. It is very soluble in water; the dissolution of the salt in water is endothermic. It is a radical initiator. It is used to etch copper on printed circuit boards as an alternative to ferric chloride solution...
(APS) (N2H8S2O8; mW: 228.2). APS is a source of free radicals and is often used as an initiator for gel formation. An alternative source of free radicals is riboflavinRiboflavin, also known as vitamin B2 or additive E101, is an easily absorbed micronutrient with a key role in maintaining health in humans and animals. It is the central component of the cofactors FAD and FMN, and is therefore required by all flavoproteins. As such, vitamin B2 is required for a...
, which generated free radicals in a photochemical reaction.
- TEMED (N, N, N', N'-tetramethylethylenediamine) (C6H16N2; mW: 116.21). TEMED stabilizes free radicals and improves polymerization. The rate of polymerisation and the properties of the resulting gel depend on the concentrations of free radicals. Increasing the amount of free radicals results in a decrease in the average polymer chain length, an increase in gel turbidity and a decrease in gel elasticity. Decreasing the amount shows the reverse effect. The lowest catalytic concentrations that will allow polymerisation in a reasonable period of time should be used. APS and TEMED are typically used at approximately equimolar concentrations in the range of 1 to 10 mM.
Chemicals for processing and visualization
The following chemicals are used for processing of the gel and the protein samples visualized in it:
- Tracking dye. As proteins are mostly colourless, their progress through the gel during electrophoresis cannot be easily followed. Anionic dyes of a known electrophoretic mobility are therefore usually included in the SDS-PAGE sample buffer. A very common tracking dye is 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...
(BPB, 3',3",5',5" tetrabromophenolsulfonphthalein). This dye is coloured at alkali and neutral pH and is a small negatively charged molecule that moves towards the anodeAn anode is an electrode through which electric current flows into a polarized electrical device. Mnemonic: ACID ....
. Being a highly mobile molecule it moves ahead of most proteins. As it reaches the anodicAn anode is an electrode through which electric current flows into a polarized electrical device. Mnemonic: ACID ....
end of the electrophoresis medium electrophoresis is stopped. It can weakly bind to some proteins and impart a blue colour.
- Loading aids. Most SDS-PAGE systems are loaded from the top into wells within the gel. To ensure that the sample sinks to the bottom of the gel, sample buffer is supplemented with additives that increase the density
The mass density or density of a material is defined as its mass per unit volume. The symbol most often used for density is ρ . In some cases , density is also defined as its weight per unit volume; although, this quantity is more properly called specific weight...
of the sample. These additives should be non-ionic and non-reactive towards proteins to avoid interfering with electrophoresis. Common additives are glycerolGlycerol is a simple polyol compound. It is a colorless, odorless, viscous liquid that is widely used in pharmaceutical formulations. Glycerol has three hydroxyl groups that are responsible for its solubility in water and its hygroscopic nature. The glycerol backbone is central to all lipids...
and sucroseSucrose 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...
.
- Coomassie Brilliant Blue R-250 (CBB)(C45H44N3NaO7S2; mW: 825.97). CBB is the most popular protein stain. It is an anionic dye, which non-specifically binds to proteins. The structure of CBB is predominantly non-polar, and it is usually used in methanolic solution acidified with acetic acid. Proteins in the gel are fixed by acetic acid and simultaneously stained. The excess dye incorporated into the gel can be removed by destaining with the same solution without the dye. The proteins are detected as blue bands on a clear background. As SDS is also anionic, it may interfere with staining process. Therefore, large volume of staining solution is recommended, at least ten times the volume of the gel.
Reducing SDS-PAGE
Besides the addition of SDS, proteins may optionally be briefly heated to near boiling in the presence of a reducing agent, such as
dithiothreitol (DTT)Dithiothreitol is the common name for a small-molecule redox reagent known as Cleland's reagent. DTT's formula is C4H10O2S2 and the molecular structure of its reduced form is shown at the right; its oxidized form is a disulfide-bonded 6-membered ring . Its name derives from the four-carbon...
or
2-mercaptoethanol (beta-mercaptoethanol/BME)2-Mercaptoethanol is the chemical compound with the formula HOCH2CH2SH. It is a hybrid of ethylene glycol, HOCH2CH2OH, and 1,2-ethanedithiol, HSCH2CH2SH...
, which further denatures the proteins by reducing disulfide linkages, thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure (oligomeric subunits). This is known as reducing SDS-PAGE, and is most commonly used.
Silver staining
In the 14th century the
silver stainSilver staining is the use of silver to selectively alter the appearance of the target.-Use in medicine:It is used to stain histologic sections. This kind of staining is important especially to show proteins and DNA. It is used to show both substances inside and outside cells...
ing technique was developed for colouring the surface of glass. It has been used extensively for this purpose since the
16th century. The colour produced by the early silver stains ranged between light yellow and an orange-red.
Camillo GolgiCamillo Golgi was an Italian physician, pathologist, scientist, and Nobel laureate.-Biography:Camillo Golgi was born in the village of Corteno, Lombardy, then part of the Austrian Empire. The village is now named Corteno Golgi in his honour. His father was a physician and district medical officer...
perfected the silver staining for the study of the
nervous systemThe nervous system is an organ system containing a network of specialized cells called neurons that coordinate the actions of an animal and transmit signals between different parts of its body. In most animals the nervous system consists of two parts, central and peripheral. The central nervous...
.
Golgi's methodGolgi's method is a nervous tissue staining technique discovered by Italian physician and scientist Camillo Golgi in 1873. It was initially named the black reaction by Golgi, but it became better known as the Golgi stain or later, Golgi method.Golgi' staining was famously used by Spanish...
stains a limited number of cells at random in their entirety. The exact chemical mechanism by which this happens is still largely unknown. Silver staining was introduced by Kerenyi and Gallyas as a sensitive procedure to detect trace amounts of proteins in
gelA 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...
s. The technique has been extended to the study of other biological macromolecules that have been separated in a variety of supports. Classical
Coomassie Brilliant BlueCoomassie Brilliant Blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. Coomassie Brilliant Blue G-250 differs from Coomassie Brilliant Blue R-250 by the addition of two...
staining can usually detect a 50 ng protein band, Silver staining increases the sensitivity typically 50 times. Many variables can influence the
colourColor or colour is the visual perceptual property corresponding in humans to the categories called red, green, blue and others. Color derives from the spectrum of light interacting in the eye with the spectral sensitivities of the light receptors...
intensity and every protein has its own staining characteristics; clean glassware, pure reagents and water of highest purity are the key points to successful staining.
Buffer systems
Most protein separations are performed using a "discontinuous" (or DISC)
bufferA buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a...
system that significantly enhances the sharpness of the bands within the gel. During electrophoresis in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all of the proteins to focus into a single sharp band. The formation of the ion gradient is achieved by choosing a pH value at which the ions of the buffer are only moderately charged compared to the SDS-coated proteins. These conditions provide an environment in which
KohlrauschFriedrich Wilhelm Georg Kohlrausch was a German physicist who investigated the conductive properties of electrolytes and contributed to knowledge of their behaviour...
reactions determine the
molar conductivityMolar conductivity is defined as the conductivity of an electrolyte solution divided by the molar concentration of the electrolyte, and so measures the efficiency with which a given electrolyte conducts electricity in solution. Its units are siemens per meter per molarity, or siemens meter-squared...
. As a result, SDS-coated proteins are concentrated to several fold in a thin zone of the order of 19 μm within a few minutes. At this stage all proteins migrate at the same migration speed by
isotachophoresisIsotachophoresis is a technique in analytical chemistry used to separate charged particles. It is a further development of electrophoresis. It is a powerful separation technique using a discontinuous electrical field to create sharp boundaries between the sample constituents.In conventional...
. This occurs in a region of the gel that has larger pores so that the gel matrix does not retard the migration during the focusing or "stacking" event. Separation of the proteins by size is achieved in the lower, "resolving" region of the gel. The resolving gel typically has a much smaller pore size, which leads to a sieving effect that now determines the electrophoretic mobility of the proteins. At the same time, the separating part of the gel also has an pH value in which the buffer ions on average carry a greater charge, causing them to "outrun" the SDS-covered proteins and eliminate the ion gradient and thereby the stacking effect.
A very widespread discontinuous buffer system is the tris-glycine or "
LaemmliUlrich K. Laemmli is a Professor in the biochemistry and molecular biology departments at University of Geneva. He is known for the refinement of SDS-PAGE, a widely-used method for separating proteins based on their electrophoretic mobility. His paper describing the method is among the most cited...
" system that stacks at a
pHIn chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
of 6.8 and resolves at a
pHIn chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
of ~8.3-9.0. A drawback of this system is that these pH values may promote
disulfideIn chemistry, a disulfide usually refers to the structural unit composed of a linked pair of sulfur atoms. Disulfide usually refer to a chemical compound that contains a disulfide bond, such as diphenyl disulfide, C6H5S-SC6H5....
bond formation between
cysteineCysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
residues in the proteins because the
pKaPKA, pKa, or other similar variations may stand for:* pKa, the symbol for the acid dissociation constant at logarithmic scale* Protein kinase A, a class of cAMP-dependent enzymes* Pi Kappa Alpha, the North-American social fraternity...
of cysteine ranges from 8-9 and because reducing agent present in the loading buffer doesn't co-migrate with the proteins. Recent advances in buffering technology alleviate this problem by resolving the proteins at a pH well below the pKa of cysteine (e.g., bis-tris, pH 6.5) and include reducing agents (e.g. sodium bisulfite) that move into the gel ahead of the proteins to maintain a reducing environment. An additional benefit of using buffers with lower pH values is that the acrylamide gel is more stable lower pH values, so the gels can be stored for long periods of time before use.
SDS gradient gel electrophoresis of proteins
As voltage is applied, the anions (and negatively charged sample molecules) migrate toward the positive electrode (anode) in the lower chamber, the leading ion is
ClChlorine is the chemical element with atomic number 17 and symbol Cl. It is the second lightest halogen, found in the periodic table in group 17. The element forms diatomic molecules under standard conditions, called dichlorine...
¯ ( high mobility and high concentration); glycinate is the trailing ion (low mobility and low concentration). SDS-protein particles do not migrate freely at the border between the Cl¯ of the gel buffer and the
GlyGlycine is an organic compound with the formula NH2CH2COOH. Having a hydrogen substituent as its 'side chain', glycine is the smallest of the 20 amino acids commonly found in proteins. Its codons are GGU, GGC, GGA, GGG cf. the genetic code.Glycine is a colourless, sweet-tasting crystalline solid...
¯ of the cathode buffer.
Friedrich KohlrauschFriedrich Wilhelm Georg Kohlrausch was a German physicist who investigated the conductive properties of electrolytes and contributed to knowledge of their behaviour...
found that
Ohm's lawOhm's law states that the current through a conductor between two points is directly proportional to the potential difference across the two points...
also applies to dissolved
electrolyteIn chemistry, an electrolyte is any substance containing free ions that make the substance electrically conductive. The most typical electrolyte is an ionic solution, but molten electrolytes and solid electrolytes are also possible....
s. Because of the voltage drop between the Cl- and Glycine-buffers, proteins are compressed (stacked) into micrometer thin layers. The boundary moves through a pore gradient and the protein stack gradually disperses due to a frictional resistance increase of the gel matrix. Stacking and unstacking occurs continuously in the gradient gel, for every protein at a different position. For a complete protein unstacking the polyacrylamide-gel concentration must exceed 16% T. The two-gel system of "Laemmli" is a simple gradient gel. The pH discontinuity of the buffers is of no significance for the separation quality, and a "stacking-gel" with a different pH is not needed.
See also
- 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...
- DNA electrophoresis
Nucleic acid electrophoresis is an analytical technique used to separate DNA or RNA fragments by size and reactivity. Nucleic acid molecules which are to be analyzed are set upon a viscous medium, the gel, where an electric field induces the nucleic acids to migrate toward the anode, due to the net...
- Eastern blotting
Eastern blotting is a biochemical technique used to analyze protein post translational modifications such as lipids and glycoconjugates. It is most often used to detect carbohydrate epitopes. Thus, Eastern blotting can be considered an extension of the biochemical technique of Western blotting...
- 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...
- 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...
- Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
- History of electrophoresis
The history of electrophoresis begins in earnest with the work of Arne Tiselius in the 1930s, and new separation processes and chemical analysis techniques based on electrophoresis continue to be developed into the 21st century...
- Isoelectric focusing
Isoelectric focusing , also known as electrofocusing, is a technique for separating different molecules by their electric charge differences...
- Isotachophoresis
Isotachophoresis is a technique in analytical chemistry used to separate charged particles. It is a further development of electrophoresis. It is a powerful separation technique using a discontinuous electrical field to create sharp boundaries between the sample constituents.In conventional...
- Native Gel Electrophoresis
- 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,...
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- Protein electrophoresis
Protein electrophoresis is a method for analysing the proteins in a fluid or an extract. The electrophoresis may be performed with a small volume of sample in a number of alternative ways with or without a supporting medium: SDS polyacrylamide gel electrophoresis Protein electrophoresis is a method...
- 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...
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- Two dimensional SDS-PAGE
- 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...
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- Zymography
Zymography is an electrophoretic technique, based on SDS-PAGE, that includes a substrate co-polymerized with the polyacrylamide gel, for the detection of enzyme activity.-Procedure:...
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