EXPERIMENTAL MOLECULAR BIOLOGY OF THE CELL

Principles of Polyacrylamide Gel Electrophoresis (PAGE)

Powerful electrophoretic techniques have been developed to separate macromolecules on the basis of molecular weight.  The mobility of a molecule in an electric 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 basis of molecular weight if, at a set voltage, a way could be found to charge these molecules to the same degree and to the same sign. Under these conditions, the mobility of the molecules would be 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 according to their molecular weights.
Polyacrylamide is formed by the polymerization of the monomer molecule-acrylamide crosslinked by N,N'-methylene-bis-acrylamide (abbreviated BIS). Free radicals generated by ammonium persulfate (APS) and a catalyst acting as an oxygen scavenger (-N,N,N',N'-tetramethylethylene diamine [TEMED]) are required to start the polymerization since acrylamide and BIS are nonreactive by themselves or when mixed together.

The distinct advantage of acrylamide gel systems is that the initial concentrations of acrylamide and BIS control the hardness and degree of crosslinking of the gel.  The hardness of a gel in turn controls the friction that macromolecules experience as they move through the gel in an electric field, and therefore affects the resolution of the components to be separated. Hard gels (12-20% acrylamide) retard the migration of large molecules more than they do 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 complex 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-PAGE)

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 charge:mass ratio for all denatured proteins. After treatment with SDS, irrespective of their native charges, all proteins acquire a high negative charge.

Denaturation of proteins is performed by heating them in a buffer containing a soluble thiol reducing agent (e.g. 2-mercaptoethanol; dithiothreitol) and SDS. Mercaptoethanol reduces all disulfide bonds of cysteine residues to free sulfhydryl groups, and heating in SDS disrupts all intra- and intermolecular protein interactions.  This treatment yields individual polypeptide chains which carry 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 modern experimental biologists. Some are mentioned below. They are employed to monitor enzyme purification, to determine the subunit composition of oligomeric proteins, to characterize the protein components of subcellular organelles and membranes, and to assign specific proteins to specific genes by comparing protein extracts of wild-type organisms and suppressible mutants. In addition, the mobility of polypeptides in SDS-PAGE 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 accuracy of +/- 5%, quickly, cheaply and reproducibly.

Discontinuous SDS Polyacrylamide Gel Electrophoresis

Disc gels are constructed with two different acrylamide gels, one on top 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 (often called the running gel), contains a higher acrylamide concentration, or a gradient of acrylamide, strongly buffered at pH 9.0. Both gels can be cast as tubes in glass or plastic cylinders (tube gels), or as thin slabs within glass plates, an arrangement which improves resolution considerably, and which makes it possible to analyze and compare many protein samples at once, and on the same gel (slab gels). Today, you will be constructing and running slab gels.

The ionic strength discontinuity 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 within the stacking gel as a rather long liquid column (0.2-0.5 cm) depending on the amount and the thickness of the gel or tube. The protein sample contains glycerol or sucrose so that it can be overlaid with a running buffer. This buffer is called the running buffer, and the arrangement is such that the top and bottom of the gel are in running buffer to make a circuit.

As current is applied, the proteins start to migrate downward through the stacking gel toward the positive pole, since they are negatively charged by the bound SDS. Since the stacking gel is very loose, low and average molecular weight proteins are not 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 electric field V/cm) to make proteins move faster than in the running gel (high ionic strength, lower resistance, hence lower electric field, V/cm). Remember that applied voltage results in current flow in the gel through the migration of ions. Hence low ionic strength means high resistance because fewer ions are present to dissipate the voltage and the electric field (V/cm) is increased causing the highly polyanionic proteins to migrate rapidly.

The rapid migration of proteins through the stacking gel causes them to accumulate and stack as a very thin zone at the stacking gel/running gel boundary, and most importantly, since the 4-5% stacking gel affects the mobility of the large components only slightly, the stack is arranged in order of mobility of the proteins in the mixture. This stacking effect results in superior resolution within the running gel, where polypeptides enter and migrate much more slowly, according to their size and shape.

In all gel systems, a tracking dye (usually Bromophenol blue) is introduced with the protein sample to determine the time at which the operation should be stopped. Bromophenol blue is a small 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 sure 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 Blue. Coomassie blue binds strongly to all proteins. Unbound dye is removed by extensive washing of the gel. Blue protein bands can thereafter be located and quantified since the amount of bound dye is proportional to protein content. Stained gels can be dried and preserved, photographed or scanned with a recording densitometer to measure the intensity of the color in each protein band. 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 will do today, the slabs of acrylamide are removed from the support glass plates and dried on filter paper. A piece of X-ray film is placed and clamped tight over the dried slab in a light-proof box. The X-ray film is exposed by the radioactivity in the protein bands and, after developing, dark spots or bands can be seen on the film. These dark bands can in turn be quantified since their intensity is proportional to the amount of radioactivity and hence to protein content.