Most protein separations are performed using a "discontinuous" (or DISC)
buffer system that significantly enhances the sharpness of the bands within the gel. During electrophoresis in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all of the proteins to focus into a single sharp band. The formation of the ion gradient is achieved by choosing a pH value at which the ions of the buffer are only moderately charged compared to the SDS-coated proteins. These conditions provide an environment in which
Kohlrausch's reactions determine the
molar conductivity. As a result, SDS-coated proteins are concentrated to several fold in a thin zone of the order of 19 μm within a few minutes. At this stage all proteins migrate at the same migration speed by
isotachophoresis. This occurs in a region of the gel that has larger pores so that the gel matrix does not retard the migration during the focusing or "stacking" event. Separation of the proteins by size is achieved in the lower, "resolving" region of the gel. The resolving gel typically has a much smaller pore size, which leads to a sieving effect that now determines the electrophoretic mobility of the proteins. At the same time, the separating part of the gel also has a pH value in which the buffer ions on average carry a greater charge, causing them to "outrun" the SDS-covered proteins and eliminate the ion gradient and thereby the stacking effect. A very widespread discontinuous buffer system is the tris-glycine or "
Laemmli" system that stacks at a
pH of 6.8 and resolves at a
pH of ~8.3-9.0. A drawback of this system is that these pH values may promote
disulfide bond formation between
cysteine residues in the proteins because the
pKa of cysteine ranges from 8-9 and because reducing agent present in the loading buffer doesn't co-migrate with the proteins. Recent advances in buffering technology alleviate this problem by resolving the proteins at a pH well below the pKa of cysteine (e.g.,
bis-tris, pH 6.5) and include reducing agents (e.g. sodium bisulfite) that move into the gel ahead of the proteins to maintain a reducing environment. An additional benefit of using buffers with lower pH values is that the acrylamide gel is more stable at lower pH values, so the gels can be stored for long periods of time before use.
SDS gradient gel electrophoresis of proteins As voltage is applied, the anions (and negatively charged sample molecules) migrate toward the positive electrode (anode) in the lower chamber, the leading ion is
Cl− ( high mobility and high concentration); glycinate is the trailing ion (low mobility and low concentration). SDS-protein particles do not migrate freely at the border between the Cl− of the gel buffer and the
Gly− of the cathode buffer.
Friedrich Kohlrausch found that
Ohm's law also applies to dissolved
electrolytes. Because of the voltage drop between the Cl− and Glycine-buffers, proteins are compressed (stacked) into micrometer thin layers. The boundary moves through a pore gradient and the protein stack gradually disperses due to a frictional resistance increase of the gel matrix. Stacking and unstacking occurs continuously in the gradient gel, for every protein at a different position. For a complete protein unstacking the polyacrylamide-gel concentration must exceed 16% T. The two-gel system of "Laemmli" is a simple gradient gel. The pH discontinuity of the buffers is of no significance for the separation quality, and a "stacking-gel" with a different pH is not needed. ==Visualization==