What Is 2D Gel Electrophoresis?

Two-dimensional (2D) gel electrophoresis is a method scientists use to take apart and analyze protein mixtures by first separating bands of proteins along two different axes. It is a technique that is most often used when dealing with complicated or thick mixtures of proteins, often with overlapping sizes of proteins. 2D gel electrophoresis differs from one-dimensional gel electrophoresis because the former method employs separation of proteins based on two different protein characteristics, whereas one-dimensional gel electrophoresis usually separates proteins based on a single characteristic, like protein size.

Gel electrophoresis is often used in research to separate molecules by making use of the fact that many biological molecules, like DNA, have an electrical charge. This fact can be used to the advantage of scientists because charged molecules can be separated by size through a porous substrate like agarose by applying a voltage gradient across a porous, ionic gel. Over time, molecules will pass through this gel, toward the charge that is opposite to the natural charge of the molecule. Small molecules and heavily charged molecules both move faster than large molecules or proteins that are weakly charged. In 2D gel electrophoresis, after separating proteins through a single characteristic, which creates a gradient, a gel can then be turned sideways to separate those previously resultant bands based on a second characteristic.

2D gel electrophoresis often employs molecular charges of proteins as one characteristic by which protein aggregates can be separated into single component proteins. Protein mass is another common characteristic used to separate proteins in 2D gel electrophoresis. After proteins are run on a gel through a single lane in one-dimensional gel electrophoresis, that gel can then be spun in a centrifuge, pulling the heavier proteins down faster than the smaller, less massive proteins. The direction of the pull is perpendicular to the direction the proteins were drawn through the gel due to attraction to an electrical charge through a voltage gradient.

Electrophoresis has many uses in molecular studies, including separation of proteins based on synthesized characteristics, like protein tagging. Protein separation based on a characteristic mass is also used when proteins are tagged with other molecules. This technique can be used with these protein complexes because the tagged proteins will fall more readily through a gel than untagged proteins.

While many separation gels in laboratories are made of agarose, protein separation by 2D gel electrophoresis is best done on polyacrylamide gels. These types of gels are used in Western Blots and other protein-size-based assays. Agarose is more often used for separation of DNA segments.