Affinity chromatography: Theory with Classification

Theory:

1. Loading/Binding Phase:

• The sample containing the target molecule is introduced into the column packed with immobilized ligand.

• The target molecule binds to the ligand due to its specific affinity, while other molecules are washed out.

2. Washing Phase:

• To remove non-specifically bound molecules or impurities, the column is washed with a suitable buffer.

3. Elution Phase:

• The bound target molecule is eluted by disrupting the specific interaction.

• This can be achieved by introducing a solution with high ionic strength, a change in pH, or a competing ligand.

4. Regeneration Phase:

• The column is returned to its original state to be used again.

• This might involve washing away the eluted target molecule and any leftover elution agents.

Classification of Affinity Chromatography:

Here's a colored diagram illustrating the classification of Affinity Chromatography:

1. Based on Type of Interaction:

1. Enzyme and Substrate/Inhibitor Chromatography: Where enzymes are attached to the resin and their substrates/inhibitors are separated based on their binding affinities.

2. Antigen-Antibody Chromatography: Antibodies (or antigens) are immobilized and used to capture antigens (or antibodies) from a mixture.

3. Receptor-Ligand Chromatography: Receptors bound to the column capture specific ligands.

2. Based on Method of Elution:

1. Biospecific Elution: Using agents that specifically compete or interfere with the target molecule's binding.

2. Non-specific Elution: Altering conditions like pH or ionic strength that affect all bound molecules.

3. Specialized Types:

1. Immunoaffinity Chromatography: Relies on the very specific binding of antibodies to their antigens.

2. Metal Chelate Chromatography: Proteins with a specific affinity for certain metals are captured on a column with metal ions.

3. Dye-Ligand Chromatography: Uses reactive dyes as ligands to bind proteins that recognize and bind to these dyes.