Title: Learn: Acrylamide Percentage Affects Separation
Course: BTEC-2020-301-302-303-F21 > Section: Protein Gels

Module2 Acrylamide Percentage Affects Separation

SDS-PAGE gels can be made (or purchased) with a variety of percentages of acrylamide. In the image below, you can see how acrylamide concentrations from 5% to 18% in the separating gel affect separation of different sized proteins. The goal is to have the proteins of interest migrate to the bottom half of the gel. Sometimes, a single gel percentage cannot provide adequate separation of a particular mixture of proteins. In this case, a gradient gel, in which the concentration starts low at the top of the gel, and gets higher toward the bottom, may be useful. Molecules with a range of sizes can be separated on gradient gels because the larger pore sizes allow resolution of larger molecules, while the decreasing pore sizes toward the bottom of the gel restrict excessive separation of small molecules. It is possible to pour gradient gels yourself, but they are usually purchased pre-made.

Different percentages of acrylamide can be used in the separating gel to optimize resolution of your sample. In general, higher percentages are better for resolving small proteins, and lower percentages are better for resolving large proteins.Gradient gels, with a low percentage of acrylamide at the top that steadily increases toward the bottom, can separate proteins with a wide range of sizes.