Полиакриламидтік гель электрофорезі: Қағидалары және қолданылуы

Analytical technique

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. Hydration of acrylonitrile results in formation of acrylamide molecules (|C3H5NO) by nitrile hydratase. 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. 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. 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. 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. 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.

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. A source of free radicals and a stabilizer, such as ammonium persulfate and TEMED are added to initiate polymerization. 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). 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%. 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.

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. 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. 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.

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.

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 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.

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). 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, a neurotoxin, and a reproductive toxin. 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. Ethidium bromide binds nucleic acid chains through the process of Intercalation. 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. Silver staining was introduced by Kerenyi and Gallyas as a sensitive procedure to detect trace amounts of proteins in gels. The technique has been extended to the study of other biological macromolecules that have been separated in a variety of supports. 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. 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. 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. 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.