The different buffers for stacking and resolving gel (discontinuous electrophoresis) are also used with gradient gels, to obtain narrow bands at the border between both gels. The acrylamide gradient in the resolving gel reduces diffusional band spreading. Leading molecules are slowed down by the higher concentration, lagging molecules accelerated by the lower. In addition, the molecular mass range of gradient gels is larger, at the expense of bands being closer together, i.e., the mass resolution is lower.

David Ruiz Pardo

In a traditional SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) system, two separate gels are used: a stacking gel and a resolving gel. The stacking gel typically has a lower acrylamide concentration and a higher pH (around 6.8) compared to the resolving gel, which has a higher acrylamide concentration and a lower pH (around 8.8). This two-gel system helps to improve the resolution and separation of protein bands during electrophoresis.

In the case of a gradient polyacrylamide gel, there is a single gel with a continuous gradient of acrylamide concentration, typically ranging from low to high acrylamide concentrations. This gradient allows for the separation of proteins based on their size more efficiently than a single fixed-concentration gel.

Although there is no distinct stacking gel and resolving gel in a gradient gel, the gradient itself serves a similar purpose. It creates a gradual change in acrylamide concentration and pH along the gel, resulting in a continuous transition from a stacking-like environment to a resolving-like environment. This gradient enables the proteins to experience a range of pore sizes, leading to effective stacking and separation.

During electrophoresis, the sample is initially loaded onto the edge of the gel with the lower acrylamide concentration, which acts as the stacking region. Here, the proteins encounter a less dense gel matrix, allowing them to concentrate into a narrow band. As the proteins migrate into the resolving region, the acrylamide concentration increases, creating a tighter and denser matrix that facilitates separation based on molecular weight.

Overall, the gradient polyacrylamide gel provides a continuous environment that combines the stacking and resolving functions into a single gel, leading to improved resolution and separation of proteins based on size.

Thanks. Your answers were really helpful. Do gradient polyacrylamide gels also have a pH gradient? Can someone please suggest literature on the matter?

No, gradient polyacrylamide gels do not typically have a pH gradient. The term "gradient" in this context refers to the concentration gradient of acrylamide within the gel matrix, which is used to separate molecules based on their size or charge.

Deepdarshan Urs : It is certainly possible to use continuous electrophoresis (only one buffer system) with gradient gels, and if continuous electrophoresis must be used, then it is suggested to at least use gradient gels to limit bandwidth.

However, the much more common application of gradient gels is with discontinuous (DISC) electrophoresis, that is, with two buffer systems. One is for the low percentage stacking gel (say, 4% T), the other for the gradient separating gel (say, 5-15% T). The two buffer systems must be matched for optimal stacking, as in the commonly used Ornstein-Davis system (DOI:10.1111/j.1749-6632.1964.tb14207.x and DOI:10.1111/j.1749-6632.1964.tb14213.x, more commonly known as Laemli system DOI:10.1038/227680a0).

David Ruiz Pardo : Stacking and separating gels usually have different pH, but the pH in the separating gel is constant even in gradient gels. Separating gels with pH-gradients are used in isoelectric focussing (IEF), but these have constant %T. Remember: it is usually best to change only one parameter at a time, if you change several you later don't know which one was important.

Thanks to all. This was really helpful.

Best wishes to everyone.