2D gel electrophoresis separates protein mixtures along two different axes, often used for complicated or thick protein mixes. The technique employs protein separation based on two different protein characteristics, unlike one-dimensional gel electrophoresis. Gel electrophoresis separates molecules by size across a porous substrate, often using molecular charges or mass as a feature. Polyacrylamide gels are best for protein separation, while agarose is often used for DNA segments.
Two-dimensional (2D) gel electrophoresis is a method used by scientists to disassemble and analyze protein mixtures by first separating the protein bands along two different axes. It’s a technique that’s most often used when dealing with complicated or thick protein mixes, often with overlapping protein sizes. 2D gel electrophoresis differs from one-dimensional gel electrophoresis because the former method employs protein separation based on two different protein characteristics, while one-dimensional gel electrophoresis usually separates proteins based on one single feature, such as protein size.
Gel electrophoresis is often used in research to separate molecules by taking advantage of the fact that many biological molecules, such as DNA, have an electrical charge. This fact can be used to the advantage of scientists because charged molecules can be separated by size across a porous substrate such as agarose by applying a voltage gradient across a porous ion gel. Over time, molecules will pass through this gel, toward the opposite charge to the molecule’s natural charge. Both small and heavily charged molecules move faster than large molecules or weakly charged proteins. In 2D gel electrophoresis, after separating proteins across a single feature, which creates a gradient, a gel can then be rotated sideways to separate those previously resulting bands based on a second feature.
2D gel electrophoresis often employs the molecular charges of proteins as a feature 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 have been flown on a gel through a single lane in one-dimensional gel electrophoresis, that gel can then be spun in a centrifuge, pulling heavier proteins down faster than smaller proteins and less massive. The direction of attraction is perpendicular to the direction the proteins were pulled through the gel due to attraction towards an electric charge across a voltage gradient.
Electrophoresis has many uses in molecular studies, including the separation of proteins based on synthesized features, such as protein labeling. Separation of proteins 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 tagged proteins will fall through a gel more easily than untagged proteins.
While many separation gels in laboratories are made from 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 most often used for the separation of DNA segments.
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