Polyacrylamide gel electrophoresis

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Picture of an SDS-PAGE. The molecular markers (ladder) are in the left lane SDS-PAGE.jpg
Picture of an SDS-PAGE. The molecular markers (ladder) are in the left lane

Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation, and charge of the molecule. Polyacrylamide gel electrophoresis is a powerful tool used to analyze RNA samples. When polyacrylamide gel is denatured after electrophoresis, it provides information on the sample composition of the RNA species. [1]

Contents

Hydration of acrylonitrile results in formation of acrylamide molecules (C3H5NO) by nitrile hydratase. [2] Acrylamide monomer is in a powder state before addition of water. Acrylamide is toxic to the human nervous system, therefore all safety measures must be followed when working with it. Acrylamide is soluble in water and upon addition of free-radical initiators it polymerizes resulting in formation of polyacrylamide. [2] It is useful to make polyacrylamide gel via acrylamide hydration because pore size can be regulated. Increased concentrations of acrylamide result in decreased pore size after polymerization. Polyacrylamide gel with small pores helps to examine smaller molecules better since the small molecules can enter the pores and travel through the gel while large molecules get trapped at the pore openings.

As with all forms of gel electrophoresis, molecules may be run in their native state, preserving the molecules' higher-order structure. This method is called native-PAGE. Alternatively, a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured molecule whose mobility depends only on its length (because the protein-SDS complexes all have a similar mass-to-charge ratio). This procedure is called SDS-PAGE. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a method of separating molecules based on the difference of their molecular weight. At the pH at which gel electrophoresis is carried out the SDS molecules are negatively charged and bind to proteins in a set ratio, approximately one molecule of SDS for every 2 amino acids. [3] : 164–79  In this way, the detergent provides all proteins with a uniform charge-to-mass ratio. By binding to the proteins the detergent destroys their secondary, tertiary and/or quaternary structure denaturing them and turning them into negatively charged linear polypeptide chains. When subjected to an electric field in PAGE, the negatively charged polypeptide chains travel toward the anode with different mobility. Their mobility, or the distance traveled by molecules, is inversely proportional to the logarithm of their molecular weight. [4] By comparing the relative ratio of the distance traveled by each protein to the length of the gel (Rf) one can make conclusions about the relative molecular weight of the proteins, where the length of the gel is determined by the distance traveled by a small molecule like a tracking dye. [5]

For nucleic acids, urea is the most commonly used denaturant. For proteins, sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein samples to coat proteins in order to impart two negative charges (from every SDS molecule) to every two amino acids of the denatured protein. [3] : 161–3  2-Mercaptoethanol may also be used to disrupt the disulfide bonds found between the protein complexes, which helps further denature the protein. In most proteins, the binding of SDS to the polypeptide chains impart an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. Proteins that have a greater hydrophobic content – for instance, many membrane proteins, and those that interact with surfactants in their native environment – are intrinsically harder to treat accurately using this method, due to the greater variability in the ratio of bound SDS. [6] Procedurally, using both Native and SDS-PAGE together can be used to purify and to separate the various subunits of the protein. Native-PAGE keeps the oligomeric form intact and will show a band on the gel that is representative of the level of activity. SDS-PAGE will denature and separate the oligomeric form into its monomers, showing bands that are representative of their molecular weights. These bands can be used to identify and assess the purity of the protein. [3] : 161–3 

Procedure

Sample preparation

Samples may be any material containing proteins or nucleic acids. These may be biologically derived, for example from prokaryotic or eukaryotic cells, tissues, viruses, environmental samples, or purified proteins. In the case of solid tissues or cells, these are often first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), by sonicator or by using cycling of high pressure, and a combination of biochemical and mechanical techniques – including various types of filtration and centrifugation – may be used to separate different cell compartments and organelles prior to electrophoresis. Synthetic biomolecules such as oligonucleotides may also be used as analytes.

Reduction of a typical disulfide bond by DTT via two sequential thiol-disulfide exchange reactions. Disulfide reduction by DTT-2.png
Reduction of a typical disulfide bond by DTT via two sequential thiol-disulfide exchange reactions.

The sample to analyze is optionally mixed with a chemical denaturant if so desired, usually SDS for proteins or urea for nucleic acids. SDS is an anionic detergent that denatures secondary and non–disulfide–linked tertiary structures, and additionally applies a negative charge to each protein in proportion to its mass. Urea breaks the hydrogen bonds between the base pairs of the nucleic acid, causing the constituent strands to separate. Heating the samples to at least 60 °C further promotes denaturation. [7] [8] [9] [10]

In addition to SDS, proteins may optionally be briefly heated to near boiling in the presence of a reducing agent, such as dithiothreitol (DTT) or 2-mercaptoethanol (beta-mercaptoethanol/BME), 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.

A tracking dye may be added to the solution. This typically has a higher electrophoretic mobility than the analytes to allow the experimenter to track the progress of the solution through the gel during the electrophoretic run.

SDS-PAGE sample.png

Preparing acrylamide gels

The gels typically consist of acrylamide, bisacrylamide, the optional denaturant (SDS or urea), and a buffer with an adjusted pH. The solution may be degassed under a vacuum to prevent the formation of air bubbles during polymerization. Alternatively, butanol may be added to the resolving gel (for proteins) after it is poured, as butanol removes bubbles and makes the surface smooth. [11] A source of free radicals and a stabilizer, such as ammonium persulfate and TEMED are added to initiate polymerization. [12] The polymerization reaction creates a gel because of the added bisacrylamide, which can form cross-links between two acrylamide molecules. The ratio of bisacrylamide to acrylamide can be varied for special purposes, but is generally about 1 part in 35. 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 molecules, while much higher percentages of acrylamide are needed to resolve smaller proteins. The average pore diameter of polyacrylamide gels is determined by the total concentration of acrylamides (% T with T = Total concentration of acrylamide and bisacrylamide) and the concentration of the cross-linker bisacrylamide (%C with C = bisacrylamide concentration). [13] The pore size is reduced reciprocally to the %T. Concerning %C, a concentration of 5% produces the smallest pores, since the influence of bisacrylamide on the pore size has a parabola-shape with a vertex at 5%.

SDS-PAGE acrylamide stock.png

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.

SDS-PAGE Acrylamide gel.png

Electrophoresis

Various buffer systems are used in 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. [9] [14] [15]

SDS-PAGE Buffers.png
SDS-PAGE Electrophoresis.png

An electric field is applied across the gel, causing the negatively charged proteins or nucleic acids to migrate across the gel away from the negative electrode (which is the cathode being that this is an electrolytic rather than galvanic cell) and towards the positive electrode (the anode). Depending on their size, each biomolecule moves differently through the gel matrix: small molecules more easily fit through the pores in the gel, while larger ones have more difficulty. The gel is run usually for a few hours, though this depends on the voltage applied across the gel; migration occurs more quickly at higher voltages, but these results are typically less accurate than at those at lower voltages. After the set amount of time, the biomolecules have migrated different distances based on their size. Smaller biomolecules travel farther down the gel, while larger ones remain closer to the point of origin. Biomolecules may therefore be separated roughly according to size, which depends mainly on molecular weight under denaturing conditions, but also depends on higher-order conformation under native conditions. The gel mobility is defined as the rate of migration traveled with a voltage gradient of 1V/cm and has units of cm2/sec/V. [3] : 161–3  For analytical purposes, the relative mobility of biomolecules, Rf, the ratio of the distance the molecule traveled on the gel to the total travel distance of a tracking dye is plotted versus the molecular weight of the molecule (or sometimes the log of MW, or rather the Mr, molecular radius). Such typically linear plots represent the standard markers or calibration curves that are widely used for the quantitative estimation of a variety of biomolecular sizes. [3] : 161–3 

Certain glycoproteins, however, behave anomalously on SDS gels. Additionally, the analysis of larger proteins ranging from 250,000 to 600,000 Da is also reported to be problematic due to the fact that such polypeptides move improperly in the normally used gel systems. [16]

Further processing

Two SDS-PAGE-gels after a completed run Gel Blue Coomassie.jpg
Two SDS-PAGE-gels after a completed run

Following electrophoresis, the gel may be stained (for proteins, most commonly with Coomassie brilliant blue R-250 or autoradiography; for nucleic acids, ethidium bromide; or for either, silver stain), allowing visualization of the separated proteins, or processed further (e.g. Western blot). After staining, different species biomolecules appear as distinct bands within the gel. It is common to run molecular weight size markers of known molecular weight in a separate lane in the gel to calibrate the gel and determine the approximate molecular mass of unknown biomolecules by comparing the distance traveled relative to the marker.

For proteins, SDS-PAGE is usually the first choice as an assay of purity due to its reliability and ease. The presence of SDS and the denaturing step make proteins separate, approximately based on size, but aberrant migration of some proteins may occur. Different proteins may also stain differently, which interferes with quantification by staining. PAGE may also be used as a preparative technique for the purification of proteins. For example, preparative native PAGE is a method for separating native metalloproteins 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. [17] [18] 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. [19] The pore size of a gel and the reproducibility in gel pore size are determined by three factors, the total amount of acrylamide present (%T) (T = Total concentration of acrylamide and bisacrylamide monomer), the amount of cross-linker (%C) (C = bisacrylamide concentration), and the time of polymerization of acrylamide (cf. QPNC-PAGE). 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 voltage 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

Polyacrylamide gels are composed of a stacking gel and separating gel. Stacking gels have a higher porosity relative to the separating gel, and allow for proteins to migrate in a concentrated area. Additionally, stacking gels usually have a pH of 6.8, since the neutral glycine molecules allow for faster protein mobility. Separating gels have a pH of 8.8, where the anionic glycine slows down the mobility of proteins. Separating gels allow for the separation of proteins and have a relatively lower porosity. Here, the proteins are separated based on size (in SDS-PAGE) and size/ charge (Native PAGE). [20]

Chemical buffer 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 counterions and thereby the resolution of the gel. The buffer should also be unreactive and not modify or 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 PAGE include Tris, Bis-Tris, or imidazole.

Counterion 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 glycine and tricine. 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 charge in the pH range. The minimum pH of this range is approximately 8.0.

Acrylamide (C3H5NO; mW: 71.08) when dissolved in water, slow, spontaneous autopolymerization 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 vinyl addition polymerisation. 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 carcinogenic, [21] a neurotoxin, and a reproductive toxin. [22] It is also essential to store acrylamide in a cool dark and dry place to reduce autopolymerisation and hydrolysis.

Bisacrylamide (N,N′-Methylenebisacrylamide) (C7H10N2O2; mW: 154.17) 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 (SDS) (C12H25NaO4S; mW: 288.38) (only used in denaturing protein gels) is a strong detergent agent used to denature native proteins to individual polypeptides. This denaturation, which is referred to as reconstructive denaturation, is not accomplished by the total linearization of the protein, but instead, through a conformational change to a combination of random coil and α helix secondary structures. [6] When a protein mixture is heated to 100 °C in presence of SDS, the detergent 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 become 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 weight. The electrophoretic mobilities of these proteins is a linear function of the logarithms 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 and molecular weight particular to its primary structure. 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.

Urea (CO(NH2)2; mW: 60.06) is a chaotropic agent that increases the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds and van der Waals forces. Macromolecular structure is dependent on the net effect of these forces, therefore it follows that an increase in chaotropic solutes denatures macromolecules,

Ammonium persulfate (APS) (N2H8S2O8; mW: 228.2) is a source of free radicals and is often used as an initiator for gel formation. An alternative source of free radicals is riboflavin, which generated free radicals in a photochemical reaction.

TEMED (N, N, N′, N′-tetramethylethylenediamine) (C6H16N2; mW: 116.21) 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 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

PAGE of rotavirus proteins stained with Coomassie blue Coomassie blue stained gel.png
PAGE of rotavirus proteins stained with Coomassie blue

The following chemicals and procedures are used for processing of the gel and the protein samples visualized in it.

Tracking dye; as proteins and nucleic acids are mostly colorless, their progress through the gel during electrophoresis cannot be easily followed. Anionic dyes of a known electrophoretic mobility are therefore usually included in the PAGE sample buffer. A very common tracking dye is Bromophenol blue (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 anode. Being a highly mobile molecule it moves ahead of most proteins. As it reaches the anodic end of the electrophoresis medium electrophoresis is stopped. It can weakly bind to some proteins and impart a blue colour. Other common tracking dyes are xylene cyanol, which has lower mobility, and Orange G, which has a higher mobility.

Loading aids; most 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 of the sample. These additives should be non-ionic and non-reactive towards proteins to avoid interfering with electrophoresis. Common additives are glycerol and sucrose.

Coomassie brilliant blue R-250 (CBB)(C45H44N3NaO7S2; mW: 825.97) 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.

Ethidium bromide (EtBr) is a popular nucleic acid stain. EtBr allows one to easily visualize DNA or RNA on a gel as EtBr fluoresces an orange color under UV light. [23] Ethidium bromide binds nucleic acid chains through the process of Intercalation. [3] While Ethidium bromide is a popular stain it is important to exercise caution when using EtBr as it is a known carcinogen. Because of this fact, many researchers opt to use stains such as SYBR Green and SYBR Safe which are safer alternatives to EtBr. [24] EtBr is used by simply adding it to the gel mixture. Once the gel has run, the gel may be viewed through the use of a photo-documentation system. [3]

Silver staining is used when more sensitive method for detection is needed, as classical Coomassie Brilliant Blue staining can usually detect a 50 ng protein band, Silver staining increases the sensitivity typically 10-100 fold more. This is based on the chemistry of photographic development. The proteins are fixed to the gel with a dilute methanol solution, then incubated with an acidic silver nitrate solution. Silver ions are reduced to their metallic form by formaldehyde at alkaline pH. An acidic solution, such as acetic acid stops development. [25] Silver staining was introduced by Kerenyi and Gallyas as a sensitive procedure to detect trace amounts of proteins in gels. [26] The technique has been extended to the study of other biological macromolecules that have been separated in a variety of supports. [27] Many variables can influence the colour 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. [28] Silver staining was developed in the 14th century 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 Golgi perfected the silver staining for the study of the nervous system. Golgi's method stains a limited number of cells at random in their entirety. [29]

Autoradiography, also used for protein band detection post gel electrophoresis, uses radioactive isotopes to label proteins, which are then detected by using X-ray film. [30]

Western blotting is a process by which proteins separated in the acrylamide gel are electrophoretically transferred to a stable, manipulable membrane such as a nitrocellulose, nylon, or PVDF membrane. It is then possible to apply immunochemical techniques to visualise the transferred proteins, as well as accurately identify relative increases or decreases of the protein of interest.

See also

Related Research Articles

<span class="mw-page-title-main">Agarose gel electrophoresis</span> Method for separation and analysis of biomolecules using agarose gel

Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The proteins may be separated by charge and/or size, and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix.

<span class="mw-page-title-main">Agarose</span> Heteropolysaccharide found in red algae

Agarose is a heteropolysaccharide, generally extracted from certain red algae. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agarose is one of the two principal components of agar, and is purified from agar by removing agar's other component, agaropectin.

<span class="mw-page-title-main">Gel electrophoresis</span> Method for separation and analysis of biomolecules

Gel electrophoresis is a method for separation and analysis of biomacromolecules and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge 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.

<span class="mw-page-title-main">Western blot</span> Analytical technique used in molecular biology

The western blot, or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecting the proteins, this technique is also utilized to visualize, distinguish, and quantify the different proteins in a complicated protein combination.

<span class="mw-page-title-main">Gel electrophoresis of nucleic acids</span>

Gel electrophoresis of nucleic acids is an analytical technique to separate DNA or RNA fragments by size and reactivity. Nucleic acid molecules are placed on a gel, where an electric field induces the nucleic acids to migrate toward the positively charged anode. The molecules separate as they travel through the gel based on the each molecule's size and shape. Longer molecules move more slowly because they the gel resists their movement more forcefully than it resists shorter molecules. After some time, the electricity is turned off and the positions of the different molecules are analyzed.

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.

<span class="mw-page-title-main">Gel electrophoresis of proteins</span> Technique for separating proteins

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, namely agarose or polyacrylamide. Variants of gel electrophoresis include SDS-PAGE, free-flow electrophoresis, electrofocusing, isotachophoresis, affinity electrophoresis, immunoelectrophoresis, counterelectrophoresis, and capillary electrophoresis. Each variant has many subtypes with individual advantages and limitations. Gel electrophoresis is often performed in combination with electroblotting or immunoblotting to give additional information about a specific protein.

<span class="mw-page-title-main">Coomassie brilliant blue</span> Chemical compound

Coomassie 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 methyl groups. The name "Coomassie" is a registered trademark of Imperial Chemical Industries.

<span class="mw-page-title-main">Temperature gradient gel electrophoresis</span>

Temperature gradient gel electrophoresis (TGGE) and denaturing gradient gel electrophoresis (DGGE) are forms of electrophoresis which use either a temperature or chemical gradient to denature the sample as it moves across an acrylamide gel. TGGE and DGGE can be applied to nucleic acids such as DNA and RNA, and proteins. TGGE relies on temperature dependent changes in structure to separate nucleic acids. DGGE separates genes of the same size based on their different denaturing ability which is determined by their base pair sequence. DGGE was the original technique, and TGGE a refinement of it.

<span class="mw-page-title-main">Southwestern blot</span> Molecular biology technique

The southwestern blot, is a lab technique that involves identifying as well as characterizing DNA-binding proteins by their ability to bind to specific oligonucleotide probes. Determination of molecular weight of proteins binding to DNA is also made possible by the technique. The name originates from a combination of ideas underlying Southern blotting and Western blotting techniques of which they detect DNA and protein respectively. Similar to other types of blotting, proteins are separated by SDS-PAGE and are subsequently transferred to nitrocellulose membranes. Thereafter southwestern blotting begins to vary with regards to procedure as since the first blotting’s, many more have been proposed and discovered with goals of enhancing results. Former protocols were hampered by the need for large amounts of proteins and their susceptibility to degradation while being isolated.

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.

TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA.

<span class="mw-page-title-main">Electrophoretic mobility shift assay</span> Technique used to study DNA and RNA

An electrophoretic mobility shift assay (EMSA) or mobility shift electrophoresis, also referred as a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common affinity electrophoresis technique used to study protein–DNA or protein–RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence, and can sometimes indicate if more than one protein molecule is involved in the binding complex. Gel shift assays are often performed in vitro concurrently with DNase footprinting, primer extension, and promoter-probe experiments when studying transcription initiation, DNA gang replication, DNA repair or RNA processing and maturation, as well as pre-mRNA splicing. Although precursors can be found in earlier literature, most current assays are based on methods described by Garner and Revzin and Fried and Crothers.

<span class="mw-page-title-main">Molecular-weight size marker</span> Set of standards

A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix. Therefore, when used in gel electrophoresis, markers effectively provide a logarithmic scale by which to estimate the size of the other fragments.

QPNC-PAGE, or QuantitativePreparativeNativeContinuousPolyacrylamideGel Electrophoresis, is a bioanalytical, one-dimensional, high-resolution and high-precision electrophoresis technique applied in biochemistry and bioinorganic chemistry to quantitatively separate proteins by isoelectric point and by continuous elution from a gel column.

<span class="mw-page-title-main">Electrophoretic color marker</span>

An electrophoretic color marker is a chemical used to monitor the progress of agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) since DNA, RNA, and most proteins are colourless. The color markers are made up of a mixture of dyes that migrate through the gel matrix alongside the sample of interest. They are typically designed to have different mobilities from the sample components and to generate colored bands that can be used to assess the migration and separation of sample components.

<span class="mw-page-title-main">Affinity electrophoresis</span>

Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. Cross electrophoresis, the first affinity electrophoresis method, was created by Nakamura et al. Enzyme-substrate complexes have been detected using cross electrophoresis. The methods include the so-called electrophoretic mobility shift assay, charge shift electrophoresis and affinity capillary electrophoresis. The methods are based on changes in the electrophoretic pattern of molecules through biospecific interaction or complex formation. The interaction or binding of a molecule, charged or uncharged, will normally change the electrophoretic properties of a molecule. Membrane proteins may be identified by a shift in mobility induced by a charged detergent. Nucleic acids or nucleic acid fragments may be characterized by their affinity to other molecules. The methods have been used for estimation of binding constants, as for instance in lectin affinity electrophoresis or characterization of molecules with specific features like glycan content or ligand binding. For enzymes and other ligand-binding proteins, one-dimensional electrophoresis similar to counter electrophoresis or to "rocket immunoelectrophoresis", affinity electrophoresis may be used as an alternative quantification of the protein. Some of the methods are similar to affinity chromatography by use of immobilized ligands.

<span class="mw-page-title-main">Discontinuous electrophoresis</span> Type of laboratory technique

Discontinuous electrophoresis is a type of polyacrylamide gel electrophoresis. It was developed by Ornstein and Davis. This method produces high resolution and good band definition. It is widely used technique for separating proteins according to size and charge.

<span class="mw-page-title-main">SDS-PAGE</span> Biochemical technique

SDS-PAGE is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. The combined use of sodium dodecyl sulfate and polyacrylamide gel eliminates the influence of structure and charge, and proteins are separated by differences in their size. At least up to 2012, the publication describing it was the most frequently cited paper by a single author, and the second most cited overall.

<i>N</i>,<i>N</i>-Diallyl-L-tartardiamide Chemical compound, polyacrylamide crosslinker

N,N′-Diallyl-L-tartardiamide (DATD) is a crosslinking agent for polyacrylamide gels, e.g., as used for SDS-PAGE. Compared to bisacrylamide gels, DATD gels have a stronger interaction with glass, and therefore are used in applications where the polyacrylamide gel acts as a "plug" structural component at the bottom of a gel electrophoresis apparatus, thereby preventing a weak discontinuous gel from sliding out from or otherwise moving within the apparatus. Unlike bisacrylamide-polyacrylamide gels, DATD-polyacrylamide gels can be conveniently dissolved using periodic acid due to the presence of viscinal diols in DATD. DATD is the slowest polyacrylamide crosslinker tested, and has can act as an inhibitor of polymerization at high concentrations.

References

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  4. ↑ Kindt T, Goldsby R, Osborne B (2007). Kuby Immunology. New York: W.H. Freeman and Company. p. 553. ISBN   978-1-4292-0211-4.
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