Affinity chromatography is a separation technique that relies on the specific binding interaction between an immobilized ligand and its binding partner. It is commonly used to purify biomolecules like proteins and enzymes. The stationary phase contains a solid support with an affinity ligand that selectively binds the target molecule. The sample is loaded and the target molecule binds while contaminants are washed away. The bound target is then eluted by changing conditions to disrupt the binding. Affinity chromatography offers high specificity and purity but can be time-consuming and require expensive ligands.

1. Affinity Chromatography
SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)

2. Introduction
 Chromatography is a powerful analytical technique used to separate a
mixture into its component parts.
 It relies on a process using the different properties that exist between the
target molecule (the molecule of interest) and its matrix, which together
make-up the sample.
 Liquid chromatography uses a liquid to carry the sample across a stationary
phase where the separation takes place.
 It is one of the main techniques used in biological sciences due to its ability
to separate samples at relatively low temperatures (as opposed to gas
chromatography which operates at high temperatures).

3. DIFFERENT TYPES OF CHROMATOGRAPHY

4. Affinity Chromatography
 Affinity chromatography is a separation method based on a specific
binding interaction between an immobilized ligand and its binding partner.
 Examples include antibody/antigen, enzyme/substrate, and
enzyme/inhibitor interactions.
 It is generally the first step, if not the only step, in a purification strategy.
 In affinity chromatography the stationary phase is critical — and is made up
of a solid support (a chemically and biologically inert medium) and a
binding agent, the affinity ligand, (that selectively binds to the target
molecule) in a column.

5. PRINCIPLE OF AFFINITY
CHROMATOGRAPHY

6. Components of Affinity
Chromatography
 Matrix: The matrix is an inert support to which a ligand can be directly or
indirectly coupled. In order to for the matrix to be effective it must have
certain characters:
1. Matrix should be chemically and physically inert.
2. It must be insoluble in solvents and buffers employed in the process.
3. It must be chemically and mechanically stable.
4. It must be easily coupled to a ligand or spacer arm onto which the ligand
can be attached.
5. It must exhibit good flow properties and have a relatively large surface
area for attachment.
6. The most useful matrix materials are agarose and polyacrylamide.

7. Components of Affinity
Chromatography
 Spacer arm: It is used to improve binding between ligand and target
molecule by overcoming any effects of steric hindrance.
 Ligand: It refers to the molecule that binds reversibly to a specific target
molecule. The ligand can be selected only after the nature of the
macromolecule to be isolated is known:
1. When a hormone receptor protein is to be purified by affinity
chromatography, the hormone itself is an ideal candidate for the ligand.
2. For antibody isolation, an antigen may be used as ligand.
3. If an enzyme is to be purified, a substrate analog, inhibitor, cofactor, or
effector may be used as the immobilized ligand.

8. Basic Steps in Affinity Chromatography
 Preparation of Column: The column is loaded with solid
support such as sepharose, agarose, cellulose etc. Ligand is
selected according to the desired isolate. Spacer arm is
attached between the ligand and solid support.
 Loading of Sample: Solution containing a mixture of
substances is poured into the elution column and allowed to
run at a controlled rate.
 Elution of Ligand-Molecule Complex: Target substance is
recovered by changing conditions to favor elution of the
bound molecules.

9. THE PROCESS OF AFFINITY CHROMATOGRAPHY - GENERALIZED

10. Mechanism of Affinity Binding
 A commonly used metaphor to illustrate affinity binding is the
lock and key analogy.
 A unique structure present on the surface of a protein is the
key that will only bind to the corresponding lock, a specific
ligand on a chromatographic support.

11. Affinity Tagged Purification
 In two-step affinity-tagged protein purification, a protein is first purified by affinity
chromatography, then desalted.
 In the first step, a recombinant protein mixture is passed over a chromatography
support containing a ligand that selectively binds proteins that contain an
affinity-tag sequence.
 Contaminants are washed away, and the bound protein is then eluted in pure
form.
 Affinity tags have different advantages.
 In immobilized metal affinity chromatography (IMAC), His binds with good
selectivity to Ni2+ or other transition metals immobilized to the ligand; the
tagged protein can be selectively eluted with imidazole.
 Tagged proteins bind to glutathione as the ligand, and are eluted with solutions
of glutathione.

12. Affinity Tagged Purification
 During the second step of desalting, affinity-purified samples can
simultaneously undergo buffer exchange to remove salts in preparation for
downstream applications.
 Desalting techniques include:
1. Size exclusion chromatography
2. Dialysis
3. Ultrafiltration
 Desalting often includes the removal not only of salt, but also of other
foreign substances, such as detergents, nucleotides, and lipids.

13. Types of Affinity Chromatography
 There are two types of affinity chromatography:
 The first method uses a naturally occurring structure or sequence of amino
acids on the protein as the binding site.
 Examples include the affinity of Affi-Gel Blue support binding for albumin’s
bilirubin-binding site and the binding of protein A in the Affi-Gel and Affi-
Prep protein A supports to the Fc region of IgG.
 An important consideration for antibody purification is to determine the
affinity of your target antibody for protein A/G chromatography media,
which varies widely.

14. Types of Affinity Chromatography
 The second method involves binding to a special amino acid sequence
engineered into the protein of interest, commonly referred to as a "tag".
 Two of the most commonly used protein tags are:
1. Polyhistidine tag, which binds to certain metal-containing complexes such
as those in Profinity IMAC resins.
2. Glutathione s-transferase (GST) sequence, which binds to glutathione,
found in Bio-Scale Mini Profinity GST media.

15. General Considerations for Affinity
Chromatography
 Since most binding interactions are based on those formed in nature, the
conditions similar to those present in most cellular organisms are usually the
best binding conditions.
 Therefore PBS (phosphate buffered saline) is often the buffer of choice.
 Conversely, conditions not normally found in vivo may alter the protein
structure enough to cause the protein to dissociate from the ligand.
 In situations where there is no recommended elution condition, a low pH
(>4) will often elute bound protein with an inherent risk of denaturation.

16. Applications of Affinity
Chromatography
 Its major application includes:
1. Separation of mixture of compounds
2. Removal of impurities or in purification process
3. In enzyme assays
4. Detection of substrates
5. Investigation of binding sites of enzymes
6. In in vitro antigen-antibody reactions
7. Detection of Single Nuceotide polymorphisms and mutations in nucleic
acids

17. Advantages of Affinity
Chromatography
 Its major advantages includes:
1. High specificity
2. Target molecules can be obtained in a highly pure state
3. Single step purification
4. The matrix can be reused rapidly
5. The matrix is a solid, can be easily washed and dried
6. Give purified product with high yield
7. Affinity chromatography can also be used to remove specific
contaminants, such as proteases

18. Limitations of Affinity Chromatography
 Its major limitations includes:
1. Time consuming method
2. More amounts of solvents are required which may be expensive
3. Intense labour
4. Non-specific adsorption cannot be totally eliminated, it can only be
minimized
5. Limited availability and high cost of immobilized ligands
6. Proteins get denatured if required pH is not adjusted