How to Read Polyacrylamide Gel Electrophoresis Results

                                    BISC411
                                     EXPERIMENTAL MOLECULAR Biology OF THE Jail cell

Principles of Polyacrylamide Gel Electrophoresis (PAGE)

Powerful electrophoretic techniques have been adult to separate macromolecules on the ground of molecular weight.  The mobility of a molecule in an electrical field is inversely proportional to molecular friction which is the result of its molecular size and shape, and directly proportional to the voltage and the charge of the molecule. Proteins could be resolved electrophoretically in a semi-solid matrix strictly on the ground of molecular weight if, at a set voltage, a way could be found to charge these molecules to the aforementioned degree and to the same sign. Under these weather condition, the mobility of the molecules would exist simply inversely proportional to their size.

It is precisely this idea which is exploited in Page to separate polypeptides according to their molecular weights. In Page, proteins charged negatively by the binding of the anionic detergent SDS (sodium dodecyl sulfate) separate within a matrix of polyacrylamide gel in an electric field co-ordinate to their molecular weights.
Polyacrylamide is formed by the polymerization of the monomer molecule-acrylamide crosslinked by Northward,North'-methylene-bis-acrylamide (abbreviated BIS). Free radicals generated past ammonium persulfate (APS) and a catalyst acting every bit an oxygen scavenger (-Northward,Northward,Due north',N'-tetramethylethylene diamine [TEMED]) are required to starting time the polymerization since acrylamide and BIS are nonreactive by themselves or when mixed together.

The singled-out advantage of acrylamide gel systems is that the initial concentrations of acrylamide and BIS control the hardness and caste of crosslinking of the gel.  The hardness of a gel in turn controls the friction that macromolecules experience every bit they move through the gel in an electric field, and therefore affects the resolution of the components to exist separated. Hard gels (12-20% acrylamide) retard the migration of large molecules more than they practice small ones. In certain cases, high concentration acrylamide gels are so tight that they exclude large molecules from entering the gel but allow the migration and resolution of low molecular weight components of a circuitous mixture. Alternatively, in a loose gel (4-8% acrylamide), high molecular weight molecules migrate much farther down the gel and, in some instances, can move right out of the matrix.

SDS Polyacrylamide Gel Electrophoresis (SDS-Folio)

Sodium dodecyl sulfate (SDS or sodium lauryl sulfate) is an anionic detergent which denatures proteins molecules without breaking peptide bonds. It binds strongly to all proteins and creates a very high and constant accuse:mass ratio for all denatured proteins. After treatment with SDS, irrespective of their native charges, all proteins acquire a loftier negative charge.

Denaturation of proteins is performed by heating them in a buffer containing a soluble thiol reducing agent (e.1000. 2-mercaptoethanol; dithiothreitol) and SDS. Mercaptoethanol reduces all disulfide bonds of cysteine residues to costless sulfhydryl groups, and heating in SDS disrupts all intra- and intermolecular poly peptide interactions.  This treatment yields individual polypeptide bondage which acquit an excess negative charge induced by the binding of the detergent, and an identical charge:mass ratio. Thereafter, the denatured proteins can be resolved electrophoretically strictly on the basis of size in a buffered polyacrylamide gel which contains SDS and thiol reducing agents.

SDS-PAGE gel systems are exceedingly useful in analyzing and resolving complex protein mixtures. Many applications and modifications of this technique are relevant to mod experimental biologists. Some are mentioned below. They are employed to monitor enzyme purification, to decide the subunit composition of oligomeric proteins, to characterize the protein components of subcellular organelles and membranes, and to assign specific proteins to specific genes past comparing protein extracts of wild-type organisms and suppressible mutants. In improver, the mobility of polypeptides in SDS-Folio gel systems is proportional to the inverse of the log of their molecular weights. This property makes it possible to measure the molecular weight of an unknown protein with an accurateness of +/- v%, quickly, cheaply and reproducibly.

Discontinuous SDS Polyacrylamide Gel Electrophoresis

Disc gels are synthetic with 2 dissimilar acrylamide gels, one on elevation of the other. The upper or stacking gel contains 4-5% acrylamide (a very loose gel) weakly buffered at pH 9.0. The lower resolving gel (oftentimes chosen the running gel), contains a higher acrylamide concentration, or a slope of acrylamide, strongly buffered at pH 9.0. Both gels can exist cast as tubes in glass or plastic cylinders (tube gels), or as sparse slabs within glass plates, an system which improves resolution considerably, and which makes information technology possible to analyze and compare many poly peptide samples at in one case, and on the same gel (slab gels). Today, you will be constructing and running slab gels.

The ionic strength aperture between the loose stacking gel and the hard running gel leads to a voltage discontinuity as current is applied. The goal of these gels is to maximize resolution of protein molecules by reducing and concentrating the sample to an ultrathin zone (1-100 nm) at the stacking gel:running gel boundary. The protein sample is applied in a well inside the stacking gel as a rather long liquid cavalcade (0.2-0.five cm) depending on the amount and the thickness of the gel or tube. The protein sample contains glycerol or sucrose and so that it can be overlaid with a running buffer. This buffer is called the running buffer, and the arrangement is such that the pinnacle and bottom of the gel are in running buffer to make a circuit.

As current is applied, the proteins first to migrate downwards through the stacking gel toward the positive pole, since they are negatively charged by the spring SDS. Since the stacking gel is very loose, low and average molecular weight proteins are non impeded in their migration and move much more quickly than in the running gel. In addition, the lower ionic strength of the stacking gel (weak buffer) creates a high electrical resistance, (i.e., a high electrical field V/cm) to make proteins movement faster than in the running gel (high ionic strength, lower resistance, hence lower electric field, V/cm). Remember that practical voltage results in electric current period in the gel through the migration of ions. Hence low ionic strength ways loftier resistance because fewer ions are nowadays to dissipate the voltage and the electric field (V/cm) is increased causing the highly polyanionic proteins to migrate apace.

The rapid migration of proteins through the stacking gel causes them to accumulate and stack every bit a very thin zone at the stacking gel/running gel purlieus, and about chiefly, since the 4-5% stacking gel affects the mobility of the large components but slightly, the stack is bundled in order of mobility of the proteins in the mixture. This stacking consequence results in superior resolution within the running gel, where polypeptides enter and migrate much more than slowly, according to their size and shape.

In all gel systems, a tracking dye (normally Bromophenol blue) is introduced with the protein sample to determine the time at which the operation should be stopped. Bromophenol blue is a pocket-sized molecule which travels essentially unimpeded just behind the ion front moving down toward the bottom of the gel. Few protein molecules travel ahead of this tracking dye. When the dye front reaches the bottom of the running gel, the current is turned off to make certain that proteins do not electrophorese out of the gel into the buffer tank.

Visualizing the Proteins

Gels are removed from tubes or from the glass plates and stained with a dye, Coomassie Brilliant Bluish. Coomassie blueish binds strongly to all proteins. Unbound dye is removed by extensive washing of the gel. Blue protein bands can thereafter exist located and quantified since the corporeality of jump dye is proportional to protein content. Stained gels can be dried and preserved, photographed or scanned with a recording densitometer to mensurate the intensity of the color in each poly peptide ring. Alternatively, if the proteins are radioactive, the protein bands can be detected by autoradiography, a technique that is widely used in modern cell and molecular biology. When gels are prepared as thin slabs to maximize resolution as you volition do today, the slabs of acrylamide are removed from the support glass plates and dried on filter newspaper. A piece of Ten-ray picture show is placed and clamped tight over the dried slab in a lite-proof box. The X-ray moving-picture show is exposed past the radioactive decay in the protein bands and, after developing, nighttime spots or bands can be seen on the picture show. These dark bands can in plough be quantified since their intensity is proportional to the amount of radioactivity and hence to protein content.

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Source: https://www1.udel.edu/biology/fschmieg/411acrylamide.htm

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