AFinity chromatography

1. Affinity
Chromatography

2. ∗1930, First developed by A.Wilhelm Tiselius,
Swedish biochemist, won the Nobel Prizein
1948
∗Used to study enzymes & other proteins
∗Relies on the affinity of various biochemical
compounds with specific properities.
Affinity History

3. ∗A method of separating biochemical
mixtures based on a highly specific
interaction between antigen & anti-
body, enzyme & substrate, or receptor
& hormone.
Affinity
Chromatography

5. Antigen Antibody
Enzyme Substrate
DNA Histon
Examples:

6. ∗Can be used to:
∗Purify & concentrate a substance from a
mixture into buffering solution.
∗Reduce the amount of a substance in
mixture
∗Purification of IgG fragments.
Uses:

7. ∗ The sample is injected into the equilibrated
affinity chromatography column.
∗ Only substance with affinity for the ligand are
retained on the column.
∗ The substance with no affinity to the ligand will
elute off.
Mechanism of Affinity
Chromatography:

10. ∗ Used in genetic engineering , ex: nucleic acid
purification.
∗ Production of vaccines , ex: anti-body purification
from blood serum.
∗ Basic metabolic research , ex: protein or enzyme
purification from cell.
Application:

13. ∗Extremely high specificity.
∗High degrees of purity can be
obtained.
∗The process is very reproducible
∗The binding sites of biological
molecules can be simply
investigated.
Advantages of Affinity
Chromatography:

14. ∗Expensive ligands.
∗Leakage of ligand.
∗Degradation of the solid support.
∗Limited lifetime.
∗Non-specific adsorption.
∗Relative low productivity.
Disadvantages:

15. ∗Hanady Khaled
∗Hend Ahmed
∗Hend Batea
∗Hend Gamal
∗Hend Hassan