Isoelectric focusing separates molecules based on their electrical charge. Proteins have a net charge determined by the pH level of their environment. Manipulating the protein’s environment with an electric field causes particles to move towards the cathode or anode, allowing for separation.
Isoelectric focusing (IEF), also called electrofocusing, is a means of separating molecules based on differences in their electrical charge. It is a type of electrophoresis, a phenomenon in which particles can be dispersed in the fluid by the application of a constant electric field. In living cells, isoelectric focusing occurs naturally, but is often recreated in the laboratory using proteins suspended in a gel.
The ability to separate molecules in isoelectric focusing relies on understanding the pH level of different proteins. Proteins can have a pH between 1 and 12. The net charge of a protein will be determined by the pH level of the local environment. For example, a protein might have a net neutral, positive, or negative charge based on the pH of its surroundings.
The net charge of any protein is the sum total of its positive and negative charges. This is determined by whether or not the amino chains that make up the protein are acidic or basic. When there are more acidic groups than basic groups, the resulting pH will be low or acidic. When there are more basic than acidic groups, the resulting pH will be high or basic. Most proteins will have a pH of 3-12, with a large concentration in the 4-7 range.
Proteins are positively charged when they are in a solution that has a lower pH value than the protein itself. They will have a negative charge when the pH value of the solution has a higher pH value than its own pH. Therefore, the pH level of the protein is called the isoelectric point of the protein. Isoelectric focusing occurs when the protein’s environment is manipulated using an electric field.
When an electric field is applied to the protein’s environment, it will affect the behavior of the particles in the protein. This means that when the protein is subjected to an environment that is at the same pH level as itself, the particles in the protein will not move. However, when the protein is subjected to an environment that has a lower pH than its own, the particles will move towards the cathode. If the protein’s environment has a higher pH than its own, the particles will move towards the anode. Isoelectric focusing requires manipulating the protein’s environment so that its particles move in the desired direction.
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