Isoelectric focusing (IEF) is a specialized electrophoresis technique used to separate proteins based on their isoelectric points (pI). The isoelectric point is defined as the specific pH at which a protein carries no net charge, resulting in a neutral state. In IEF, a stable pH gradient is established within a gel, where the pH decreases linearly from a higher value at the top to a lower value at the bottom. For instance, the pH might start at 9 at the top and decrease to 3 at the bottom, remaining constant in each section of the gel throughout the process.
As proteins migrate through the gel, they encounter different pH levels, which affect the ionization of their ionizable groups. This change in pH alters the charge of the proteins, causing them to move until they reach the region where the pH equals their pI. At this point, the protein achieves a neutral net charge and ceases to migrate in the electric field. Consequently, distinct protein bands form at various locations in the gel, corresponding to their respective pI values.
To summarize, the key concept of isoelectric focusing is that proteins will migrate through the gel until they reach the pH that matches their pI, at which point they stop moving. This technique allows for the effective separation of proteins based on their isoelectric points, facilitating further analysis and study of protein characteristics.
