The separation of proteins in the case of isoelectric focusing is based on the relative content of positive and negative charged groups.

Isoelectric focusing (IEF) is a powerful technique used to separate proteins based on their isoelectric points (pI). The pI is the pH at which the protein has no net charge. Proteins can be positively charged, negatively charged, or have no charge depending on the pH of the surrounding medium.

Principle of Isoelectric Focusing:
Isoelectric focusing is based on the principle of establishing a pH gradient in a gel matrix. This is achieved by placing a mixture of protein samples in a gel with a pH gradient. The gel is then subjected to an electric field, causing the proteins to migrate towards their respective pI points.

How does it work?
During isoelectric focusing, the proteins migrate in the gel until they reach a region with a pH equal to their pI. At this pH, the proteins have no net charge and stop migrating. This phenomenon is called "focusing" because the proteins become concentrated at their respective pI points.

Role of Charged Groups:
The separation of proteins in isoelectric focusing is based on the relative content of positive and negative charged groups present in the protein molecules. Proteins have different pI values depending on the distribution of charged groups along their polypeptide chains.

Proteins with Positive Charge:
Proteins with a higher relative content of positive charged groups will have a lower pI value. These proteins will migrate towards the negatively charged electrode until they reach a pH region equal to their pI, where they will stop.

Proteins with Negative Charge:
Proteins with a higher relative content of negative charged groups will have a higher pI value. These proteins will migrate towards the positively charged electrode until they reach a pH region equal to their pI, where they will stop.

No Charge Proteins:
Proteins with no net charge will migrate until they reach a region with a pH equal to their pI, where they will also stop.

Conclusion:
In summary, the separation of proteins in isoelectric focusing is based on the relative content of positive and negative charged groups present in the proteins. Proteins migrate in the gel until they reach a pH region equal to their pI, where they stop due to their charge distribution. This technique allows for the precise separation and analysis of proteins based on their isoelectric points.