Protein Gels and Stains Used for Protein Separation
In the figures on this page: Factors Influencing Protein Separation, some gels list a single acrylamide percentage, whereas other gels show a range of acrylamide concentrations (e.g., 4–12%). Gels that have a single acrylamide percentage are referred to as linear gels, and those with a range are referred to as gradient gels. The advantage of using a gradient gel is that it allows the separation of a broader range of proteins than does a linear gel.
Researchers occasionally refer to gels as continuous or discontinuous. A continuous gel is a gel that has been formed from a single acrylamide solution in the entire gel cassette. A discontinuous gel is actually formed from two acrylamide solutions, a small, low percentage stacking gel where the protein wells reside, and a larger portion of gel that separates the proteins. In the traditional Tris-glycine protein gel system, the proteins are stacked in the stacking gel between the highly mobile leading chloride ions (in the gel buffer) and the slower, trailing glycine ions (in the running buffer).
The reason for using the stacking gel is to improve the resolution of the bands in the gel. These stacked protein bands undergo sieving once they reach the separating gel. However, the resolution of smaller proteins (<10 kDa) is hindered by the continuous accumulation of free dodecyl sulfate (DS) ions (from the SDS in the sample and running buffers) in the stacking gel. This zone of stacked DS micelles causes mixing of the DS ions with the smaller proteins, resulting in fuzzy bands and decreased resolution. The mixing also interferes with the fixing and staining of smaller proteins.