A fixed or a straight SDS-PAGE gel consists of stacking (top) and resolving (bottom) acrylamide matrix components. The stacking component has a lower concentration of acrylamide, compared to the resolving component, which concentrates the sample before separation1. The resolving gel has a homogenous composition and uniform pore size to enable separation of proteins with similar molecular weights.
A gradient gel consists of a matrix with different concentrations of acrylamide. The pore sizes at the top of the gel are usually larger (lower concentration of acrylamide), with sizes gradually decreasing towards the bottom of the gel (higher concentration of acrylamide). Gradient gels are effective at separating proteins in a complex sample with a broad range of molecular weights. Depending upon molecular weight differences, gradient gels may be able to effectively separate phosphorylated and non- phosphorylated forms of a protein of interest.
For samples containing protein complexes, it is best to separate them on a gradient gel first and identify a narrower molecular weight range for the proteins of interest. The samples can then be electrophoresed on a fixed gel to get better resolution between similar molecular weight proteins.
Both fixed and gradient gels can be poured in the lab or alternatively purchased pre-cast. The number of wells, well volume, and type of buffering system (phosphate-buffered saline or tris-buffered saline) to be used are all important considerations for effective protein separation.