What fundamental mechanism enables affinity chromatography to selectively separate sample components?
It is based on reversible molecular interactions known as affinity or biological recognition.
What is an alternative name frequently used for affinity chromatography?
Bio-selective adsorption.
Which scientists first introduced affinity chromatography in 1968 with enzyme purification methods using immobilized substrates?
Chris Anfinsen, Meir Wilchek, and Pedro Cuatecasas.
What key property must the chromatography matrix have to function properly in affinity chromatography?
It must be chemically and physically inert to avoid unwanted interactions.
Which two materials are most commonly used as matrices in affinity chromatography?
Agarose and polyacrylamide.
In affinity chromatography, what defines a ligand?
A molecule that reversibly binds a specific target molecule.
What happens to molecules that do not interact with the immobilized ligand?
They elute early, passing through the column unretained in the void volume.
Which ligands are typically used for antibody purification?
Antigens or haptens.
What is the role of a spacer arm in affinity chromatography?
To reduce steric hindrance and improve accessible binding between ligand and target.
What is the optimal length of a spacer arm?
Between 6 and 10 carbon atoms or an equivalent distance.
What are the three main steps in the affinity chromatography procedure?
Sample loading, washing off non-specific binders, and eluting the target molecule.
How are target molecules specifically eluted from the affinity column?
By introducing a competitive ligand in the mobile phase.
Name a non-specific method used to elute bound molecules in affinity chromatography.
Altering pH, ionic strength, or buffer polarity.
According to the Renkin equation, what pore size is recommended for a protein of 60 Å diameter?
Approximately 300 Å (five times the protein diameter).
Besides covalent attachment, what is a common method to immobilize ligands onto a matrix?
Adsorption.
For antibody (IgG) purification from serum, which ligand is preferable?
Protein A or Protein G.
How is the purified protein generally released from the ligand?
By adding a competitive ligand to displace it.
Which affinity chromatography method is suited for purifying His-tagged recombinant proteins?
Immobilized Metal Ion Affinity Chromatography (IMAC).
Why is affinity chromatography favored for monoclonal antibody purification in pharmaceuticals?
It provides high selectivity and purity in a single step.
What ligand property ensures effective separation in affinity chromatography?
Weak and reversible binding to the target molecule.
Protein A and Protein G are commonly used to isolate which biomolecule?
Immunoglobulins (antibodies).
Which ligand type is used specifically in immunoaffinity chromatography?
Antibodies.
Lectin ligands recognize and bind to which biomolecules?
Specific carbohydrate structures.
What is the effect of introducing charged groups into the matrix during ligand immobilization?
It may cause nonspecific binding by ion-exchange effects.
