by K Chico, Jessica Piczon

The concentration of the gel is crucial for any gel electrophoresis experiment, and the optimal gel concentration, regardless of matrix type, for an experiment depends on the sizes of the starting proteins and desired fragments size.  

For any gel used in electrophoresis, the concentration determines the resolution of the fragmented results. One of the largest benefits of agarose gels is their ease of construction. In essence, a desired amount of agarose powder can be mixed with tris-borate-EDTA (TAE) buffer, briefly heated, and dissolved.

During the gelation process, agarose polymers associate non-covalently and form a polysaccharide web. The pore sizes of this web determine a gel's molecular sieving abilities. Though gels with 0.5-3% agarose concentration can be used, 0.7-2.0% is most common. Higher agarose concentration results in a greater resolving power, however gels that are too high in concentration are brittle and may not set evenly. Alternatively, low concentration of agarose gel can be used for isoelectric focusing, though gels that are too low in concentration are weak and may break when handled. Really, 0.7-2.0% agarose concentration leaves very little room for error, so it is critical that required concentration be determined prior to experimentation. For reference, approximate fragment resolution based on agarose concentration in agarose gels is shown below.

Polyacrylamide gels can offer a high degree of reproducibility and more precise porosity. Polyacrylamide gels are prepared by the polymerization of acrylamide, a free radical, and bis-acrylamide, a cross-linking co-monomer. Polymerization depends on the concentration of monomers and catalysts (commonly, ammonium persulfate (APS) and tetramethylethylenediamine (TEMED)) present, the reaction temperature, and the purity of the reagents. These factors must be carefully controlled as this reaction is exothermic; the formation of too much heat, too rapidly, may lead to non-uniform pores. Acrylamide concentrations of 3-30% have been reported, though 7-12% is most common. A lower acrylamide concentration will allow larger proteins to pass through the gel, while a higher acrylamide concentration does the opposite. Unlike agarose gels, acrylamide gel concentrations are easily modifiable providing the possibility for various gel conformations.

In SDS-PAGE, the gel is made of two parts, termed the stacking and resolving gels. The stacking gel is the upper portion of the gel close to the wells and is normally a concentration of around 4% acrylamide while the resolving gel located at the bottom of the assembly is usually a concentration of around 15%. In experimentation, the stacking gel concentrates the proteins into a sharp band prior to separation in the higher concentration resolving gel.

Alternatively, a gradient gel may be preferred. These gels have lower acrylamide percentages where the sample enters, which increases along the sample's path so that a broader range of protein sizes can be separated. Though the construction is timelier, polyacrylamide gradient gels allow for a broader range of protein sizes to be resolved in one gel, produce sharper bands, and better separate similar-sized proteins. For reference, approximate fragment resolution based on acrylamide concentration is shown below for double-stranded (native) and single-stranded (denatured) DNA.