PAGE gel density

The density of the polyacrylamide gel is adjusted according to the expected molecular weights of the proteins to be separated. The density comes from the polymerization reaction between acrylamide and bis-acrylamide. The bis-acrylamide forms cross links between two acrylamide molecules, meaning the more acrylamide added to the mix, a denser matrix is formed in the gel. Generally, the acrylamide concentration is varied between 5-25%. Lower percentage gels are better for separating higher molecular weights, as the matrix formed has larger pore sizes. Higher percentages of acrylamide are used for lower molecular weights as the matrix formed has smaller pore sizes.

Illustration of three gels with different concentrations of acrylamide. To the left is a gel with low acrylamide concentration, in the middle is a gel with a medium concentration and to the right is a gel with a high concentration. Proteins of different sizes are being separated in the gels, where the gel with a low concentration of acrylamide, allows all proteins to migrate to the bottom of the gel and is thereby not separated properly. In the gel with a medium concentration, all proteins have been separated by size, so that the largest ones are present at the top of the gel, the medium sized proteins in the middle of the gel and the smallest proteins in the bottom of the gel. All proteins are present at the top of the gel with a high acrylamide concentration, as the density doesn’t allow them to separate through the gel.

Figure 1. Separation of proteins in gels of different densities: Separation of proteins in a gel with a low, medium and high acrylamide concentration.