Two-dimensional (2D) electrophoresis is a powerful analytical technique that combines two distinct electrophoresis methods: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This method allows for the separation of proteins based on their isoelectric points (pI) and molecular weights, providing a more comprehensive analysis than either technique could achieve alone.
In the first dimension, isoelectric focusing separates proteins according to their pI, which is the pH at which a protein carries no net charge. A stable pH gradient is established in the IEF gel, allowing proteins to migrate until they reach the region where the pH matches their pI. Proteins with higher pI values will concentrate at the top of the gel, while those with lower pI values will settle towards the bottom.
After the IEF step, the gel is rotated 90 degrees to prepare for the second dimension, SDS-PAGE. This technique separates proteins based on their molecular weights. In SDS-PAGE, proteins are denatured and coated with sodium dodecyl sulfate, which imparts a negative charge proportional to their size. As the proteins migrate through the gel, larger proteins move more slowly and remain closer to the top, while smaller proteins travel further down the gel.
The combination of these two methods allows 2D electrophoresis to effectively distinguish proteins that share identical pIs but differ in molecular weight, as well as those that have the same molecular weight but different pIs. This dual separation capability is particularly valuable in proteomics, where complex protein mixtures need to be analyzed.
When interpreting a 2D electrophoresis gel, each spot represents a unique protein. The vertical axis corresponds to the pI, while the horizontal axis represents molecular weight. It is crucial to pay attention to the labeling of these axes, as the orientation can affect the interpretation of the results. Overall, 2D electrophoresis is an essential technique for detailed protein characterization and analysis.