The original use of polyacrylamide gels as electrophoretic separating media involved using a single gel with a uniform pH throughout. This system has only occasional use in today's laboratory and replaced by discontinuous gel electrophoresis. Discontinuous gel electrophoresis system uses a separating gel of 2-5% of acrylamide (larger pore size) with pH 6.8 while resolving gel is at 12-20% acrylamide (small pore size) with pH 8.8. As you can see the pH of the gel is discontinuous. What are the advantages of a discontinuous gel system? The main advantage is that the proteins electrophorese quickly through the stacking gel of larger pore size and "stack" at the interface of resolving gel where they experience bigger pore size. In the resolving gel proteins are separated based on mass or size. The other advantage of the discontinuous system is protein stacking by a process call isotachphoresis .

The electrophoratic buffer in cathodic chamber contains chloride ions (called the leading ions) whose electrophoretic mobility is greater than the mobility of the proteins in the sample because of highest charge density. The electrophoresis buffer contains glycine ions (called the trailing ions) whose electrophoretic mobility is less than the mobility of the proteins in the sample as only a small fraction of glycine will be in anionic form giving overall small negative charge. The net result is that the faster migrating Cl^- ions leave a zone of lower conductivity between themselves and the migrating protein. The higher voltage gradient in this zone allows the proteins to move faster and to "stack" in the zone. However, as soon as protein crossed Cl^- ion zone, it will experience zone of high conductance (low voltage) and slow down. The same phenomenon will keep Glycine behind Protein. As protein is moving faster than Glycine, it will leave a zone of low conductance (high voltage). Glycine will move fast to reach zone of high voltage but as soon as reaches zone of protein it will experience relatively low voltage and slow down. In this way protein reached interface of stacking and resolving gel stacked between Glycine and Cl^- ions. The process is called isotachphoresis. After leaving the stacking gel, the protein enters the separating gel. The separating gel has a smaller pore size and higher pH compared to the stacking gel. In the separating gel, the ionization of glycine favours formation of more anion form (Henderson-Hasselbach equation) giving net charge density on glycine higher then protein. Thus, in resolving gel Cl^- ion moves fastest, followed by Glycine. Proteins are not sandwiched between these two like stacking gel. Here proteins are separated based on mass (as charge density is identical in all protein after SDS treatment). Before loading a protein sample in polyacrylamide gel it is boiled with loading buffer which contains sodium laurlysulfate SDS (C₁₂ H₂₅ SO₄ Na), anionic surfactant and reducing agants like dithiothreitol (DTT) or 2-mercaptoethanol. Most of the proteins binds SDS with constant-weight ratio (one SDS molecule per two amino acids) leading to identical charge densities. However, unless proteins are completely unfolded SDS cannot bind uniformly to protein amino acid residues as some amino acid residues may not be accessible due to folding of protein. Thus, reducing agent, which further denatures the proteins by reducing disulfide linkages and enables SDS to bind proteins uniformly. There are two more ingredients of loading buffer Bromophenol Blue and glycerol. Glycerol increases density of loading buffer so sample can settle in loading well while Bromophenol Blue is a colored dye and indicates progression of electrophoresis. Electrophoresis is performed at constant current till bromophenol blue band reaches bottom of the gel.