Affinity chromatography is a powerful technique for purifying proteins using biological interactions like enzyme-substrate binding. It works by attaching a ligand with specific binding affinity to a stationary phase within a column. When a protein mixture is passed through, only the desired proteins that bind to the ligand will be retained while others pass through. Various methods can then be used to separate the purified proteins from the ligand, such as changing pH, salt concentration, or adding competitors. Affinity chromatography is useful for purifying molecules like antibodies, enzymes, and nucleic acids.

1. Submitted by,
NAVAMI S S
1st yr Biochemistry
Dept.of Life Sciences

2.  Affinity chromatography is a powerful technique of purifying proteins.
 Biological macromolecules, such as enzymes and other proteins, interact
with other molecules with high specificity through several different types of
bonds and interaction. Such interactions include hydrogen bonding, ionic
interaction, disulfide bridges, hydrophobic interaction, and more.
 The high selectivity of affinity chromatography is caused by allowing the
desired molecule to interact with the stationary phase and be bound within
the column in order to be separated from the undesired material which will
not interact and elute first.
 The molecules no longer needed are first washed away with a buffer while
the desired proteins are let go in the presence of the eluting solvent (or
higher salt concentration).

3. Chromatography is an important biophysical technique that enables the
separation, identification, and purification of the components of a mixture
for qualitative and quantitative analysis.It is a separation technique in which
a mobile phase carrying a mixture is caused to move in contact with a
selectively absorbent stationary phase.
Affinity chromatography is a type of liquid chromatography for the
separation, purification or specific analysis of sample components.It utilizes
the reversible biological interaction or molecular recognition called affinity
which refers to the attracting force exerted in different degrees between
atoms which cause them to remain in combination.
Example: Enzyme with an inhibitor, antigen with an antibody, etc.
× It was discovered by Pedro Cuatrecasas and Meir Wilcheck.

4. The stationary phase consists of a support medium, on which the
substrate (ligand) is bound covalently, in such a way that the
reactive groups that are essential for binding of the target
molecule are exposed.
As the crude mixture of the substances is passed through the
chromatography column, substances with binding site for the
immobilized substrate bind to the stationary phase, while all other
substances is eluted in the void volume of the column.

5.  After all the other proteins have been removed and bound, it is
now possible to bind the enzyme(s) are able to be removed in a
variety of ways:
1. by increasing the ionic strength of buffer e.g. By introducing the
sodium chloride gradient which weakens the interactions that the
enzyme has with its immobilised substrate.
2. by altering the pH in the buffer. This is thereby lessening the
interaction with the substrate and enzyme.
3. by adding a significant amount of substrate (or an analogue of the
substrate) to the buffer for elution, to ensure that there is a competing
between the immobilised and free substrates for the enzyme protein.

7. 1. Matrix
The matrix acts as an inert structure that a ligand may be connected
directly or indirectly. The most efficient matrix materials are
polyacrylamide and agarose. It is distinguished by its unique
properties like;
Doesn’t itself absorb molecules in a substantial quantity.
Ligands should be coupled without altering the binding properties.
Stability in a variety of conditions for experiments, such as both low
and high pH detergent, as well as dissociating conditions.

9. 2. Spacer arm
It prevents the ligand from attaching to the matrix, which could
interfere with its ability to bond to macromolecules.
 The optimal length is 6-10 carbon atoms or equivalent.
 Most commonly, it is utilized for smaller immobilized ligands.
Examples of Spacer arms are 1,6-diamino Hexane and 6-amino
Hexanoic Acid.

10. 3.Ligand
 The ligand is a molecule which binds reversibly with an individual
chemical or group of molecules, making it possible to purify the
sample using affinity chromatography.
 The choice of a specific ligand to be used for affinity
chromatography can be influenced by two aspects:
1. The ligand has to show an irreversible and specific binding affinity
for the targeted substance(s)
2. It should possess chemically modifiable groups that permit it to be
bonded to the matrix without damaging the binding function.

11.  Any element can be utilized as a ligand for purification of the binding partner of
its choice. A few of the most common biological interactions often used in affinity
chromatography are as follows:
1. Enzyme used for substrate analog and inhibitors, as well as cofactor.
2. Antibody used for antigen, virus, cell.
3. Lectin is a polysaccharide glycoprotein cell surface receptor cell.
4. Nucleic acid is used to complement base sequences, histones nucleic acid
polymerase and nucleic acid-binding protein.
5. Hormone vitamin, used to receptors, carriers protein.
6. Glutathione used for glutathione-S-transferase or GST fusion proteins.
7. Metal ions that are used in Poly (His) fusion proteins and native proteins that
contain histidine, cysteine or tryptophan residues on their surface.

13. Step: 1 Attach ligand to column matrix
The binding of the ligand with the matrix requires that a covalent bond is
created between both. This is accomplished through derivatization of the sugar-
based” hydroxyl groups. The substrate may not be able to access the active site
of the ligand, in the event that it is hidden inside the ligand. The majority of
ligands are connected to spacer arms that are then attached onto the matrix.
The matrix-ligand gel is loaded into an column of elution.

14. Step 2: Load Protein Mixture onto the Column
After the column is made, the mix containing isolate is then poured into the column
for elution. Gravity pulls the solution into the gel because most of the proteins don’t
attach to the ligand-matrix. If ligand that is recognized as substrate moves through
the gel it bonds to the ligand-matrix complex, stopping its flow within the gel.
Certain impurities pass through the gel because of gravity, however the majority
remain unbound in the gel column.
Step 3: Proteins Binds to the ligand
To remove these impurities which are not bound, the wash must be of high pH or
salt concentration or temperature is passed across the gel. It is crucial to make the
most powerful wash possible to ensure all impurities are eliminated. After the
impurities have been removed then the only thing left of the protein mixture must
be the specific isolates.

15. Step 4: Wash the column to remove the unwanted Materials
To finally collect an isolate that is attached to the ligand matrix in the gel second
wash is run over the column.
Step 5: Wash off the Proteins that are loosely bind
The second wash is based on the reversible properties of binding of the ligand. This
lets the bound protein separate from its ligand in the presence of the more
powerful wash.
Step 6: Elute proteins that are tightly bound to ligand and
collect purified protein and interest
The protein then has the freedom to pass through the gel before being removed.

17. 1.pH elution
 A variation in pH alters the amount of ionization by charged groups on the
ligand as well as proteins bound. The alteration could affect site of binding
directly decreasing its affinity or trigger an indirect change in affinity due to
modifications in the conformation.
 A gradual decrease by a step in pH can be the most popular method of
eluting bound substances. Chemical stabilities of the matrix, the ligand, and
the target protein determines the limits of pH which can be used. If a low pH
is required, then collect the fractions into neutralization buffers like 1 M Tris-
HCl .pH 9 (60-200 mg ul for each ml fraction eluted) to bring the fraction
back to an equilibrium pH. The column should be returned in the neutral pH
immediately.

18. 2. Ionic strength elution
 The exact mechanism behind elimination through changes in the strength of ionic
depends on the specific relationship between the ligand and the target protein.
This is a moderate elimination using a buffer that has higher Ionic strength (usually
NaCl), applied in a linear manner.
3. Competitive elution
 They are typically used to distinguish substances in an individual medium or when
the attraction of the target protein interactions is quite high. The eluting agent is
competing with the protein of interest or for binding to the binding ligand.
Substances can be eluted by the concentration gradient of one Eluent, or by pulse
elimination.
 In the case of competitive elution the concentration of the competing compound
should be equal to that of the ligand that is coupled. If, however, the competing
compound is able to bind more easily than the ligand targeted molecule, then
use an amount that is ten times greater than the ligand.

19. 4. Reduced polarity of eluent
 Conditions are employed to reduce the polarity of the eluent and allow for elution
but not inactivating the substances that are eluted.
 Dioxane (up to 10 10%) as well as Ethylene glycol (up to 50 percent) are common
to this kind of liquid eluent.
5. Chaotropic eluents
 If other methods of elution fail the deforming buffers which alter the protein’s
structure can be employed.
e.g. Chaotropic agents like the guanidine hydrochloride and urea.
 Avoid chaotropes whenever possible as they can cause to cause denature of the
protein eluted.

20. 1.Lectin Affinity Chromatography
 Purification of glycoproteins, specifically the membrane-receptor proteins.
 Lectins are a class of proteins made by animals and plants that are able to
bind glycoproteins and carbohydrates.
 Useful to separate cells into different types, making use of saccharide
component of their outer membranes.
 The most commonly used lectins include: ConcanavalinA Soyabean lectin,
etc..

21. 2. Immuno Affinity chromatography
 It is used in the isolation and elimination of a variety of proteins such as antigens,
membrane proteins that are of viral origin.
 It is used to purify antibodies.
 Ligands used include the Protein A as well as Protein G.
3. Metal Chelate Chromatography
 Special type of chromatography which immobilised metal ions such as Cu2+ Zn2+ ,
Mn2+, Ni2+ etc. are employed.
 It is used to purify proteins that contain imidazole groups or indole groups.
 Commonly metal ions are immobilised by attachment to an imino-diacetate or
tris(carboxymethyl)ethylenediamine substituted agarose.

22. 4. Dye Ligand Chromatography
 Utilizes a variety of triazine dyes for ligands.
 The most popular color is Cibracron Blue F3G-A.
 It is used to purify interferons and lipoproteins as well as factors that cause
coagulation, etc.
5. Covalent chromatography
 Specially designed to separate proteins containing thiol
 The most frequently used ligand an adiquate 2′-pyridyl group
 It is used to purify many proteins, however its application is restricted due to its
price and difficult regeneration.

23. TYPES OF AFFINITY MEDIA USED IN AFFINITY
CHROMATOGRAPHY
A variety of affinity media are available to serve a range of applications. Briefly, they
are (generalized) activated/functionalized that work as a functional spacer, support
matrix, and eliminates handling of toxic reagents.
 Amino acid media: It is used in conjunction with a variety of proteins from serum
enzymes, and peptides in addition to dsDNA and rRNA.
 Avidin biotin media: Avidin biotin media is used to purify the process of
biotin/avidin, as well as their derivatives.
 Carbohydrate bonding is most often used with glycoproteins or any other
carbohydrate-containing substance; carbohydrate is used with lectins,
glycoproteins, or any other carbohydrate metabolite protein.

24.  The dye ligand medium is nonspecific however it mimics biological substrates as
well as proteins.
 Hydrophobic interaction medium are frequently employed to attack free
carboxyl groups and proteins.
 Immunoaffinity media uses antigens’ as well as antibodies that have high
specificity to differentiate immobilized metal affinity chromatography . It uses
interactions between proteins and metal ions (usually specifically labeled) to
separate. Nucleotide/coenzyme which helps separate dehydrogenases, kinases
and transaminases.
 Speciality media are made for specific classes or types of co enzyme. This kind of
media can only function to isolate a particular type of protein, or coenzyme.

25. WEAK AFFINITY CHROMATOGRAPHY
 WAC is a weak affinity technique. (WAC) is an affinity chromatography
technique used for testing affinity for drug development.
 It is an affinity-based technique for liquid chromatography which separates
chemical compounds according to their weak affinities with the immobilized
object.
 The greater affinity a compound has with the target the longer it will remain
within the separator and this is measured as a longer retention time.
 The measurement of affinity and rank of affinity are accomplished by
processing the retention times of the compounds being studied.

26.  Affinity chromatography is a part of a wider set of chemoproteomics
techniques for the purpose of identifying drug targets.
 The WAC technology has been demonstrated against various protein
targets , including proteases chaperones, kinases, along with protein-
protein interaction (PPI) specific targets.
 WAC has been proven to be more efficient than traditional methods for
screening based on fragments.

27.  It is used to isolate and purification of all biological macromolecules.
 It is used to purify nucleic acid, antibodies, enzymes etc
 To decrease the amount of substance present in a mix.
 Utilized for Genetic Engineering for nucleic acid purification
 Utilized for the Production of Vaccines – antibody purification from blood
serum
 It is used for Basic Metabolic Research such as the purification of enzymes or
proteins from cells free extracts.
 Affinity chromatography also serves to get rid of particular contaminants, like
that of Benzamidine. Sepharose(tm) 6 Fast Flow removes serine proteases
like the Factor Xa and thrombin.

28.  Antibodies can be immobilized by both covalent and adsorption methods.
Random covalent immobilization methods generally link antibodies to the
solid support via their free amine groups using cyanogen bromide, N-
hydroxysuccinimide, N,N’-carbonyldiimidazole, tresyl chloride, or tosyl
chloride.
 Alternatively, free amine groups can react with aldehyde or free epoxy
groups on an activated support. As these are random immobilization
methods, the antibody binding sites may be blocked due to improper
orientation, multi-site attachment or steric hindrance. Site-specific covalent
immobilization of antibodies can be achieved by converting the
carbohydrate residues located in the Fc region of the antibody to produce
aldehyde residues which can react with amine or hydrazide supports.
1. IMMUNOGLOBULINS PURIFICATION
(antibody immobilization)

29. 2. RECOMBINANT
TAGGED PROTEINS
 Purification of proteins can be easier and
simpler if the protein of interest is
tagged with a known sequence
commonly referred to as a tag.
 This tag can range from a short
sequence of amino acids to entire
domains or even whole proteins.
 Tags can act both as a marker for
protein expression and to help facilitate
protein purification.
3. PROTEIN
A, G, & L PURIFICATION
• Proteins A, G, and L are native
or recombinant proteins of
microbial origin which bind
specifically to
immunoglobulins including
immunoglobulin G (IgG).
• IgG represents 80% of serum
immunoglobulins.
• The most popular matrixes or
supports for affinity
applications which utilize
protein A, G, or L is beaded

30. 3A. GST TAGGED PURIFICATION
 Separation and purifcation of GST-tagged proteins is possible since the GST
tag is capable of binding its substrate, glutathione (tripeptide, Glu-Cys-Gly).
 When glutathione is reduced (GSH), it can be immobilized onto a solid
support through its sulfhydryl group. This property can be used to crosslink
glutathione with agarose beads and, thus, can be used to capture pure GST or
GST-tagged proteins via the enzyme-substrate binding reaction.
 Binding is most efficient near neutral physiological conditions (pH 7.5) using
Tris saline buffer and mild conditions to preserve the structure and enzymatic
function of GST.
 As a result of the potential for permanent denaturation, denaturing elution
conditions are not compatible with GST purification. In addition, upon
denaturation or reduction, the structure of the GST fusion tag often degrades.

31. 3B. HIS-TAGGED
PROTEIN
PURIFICATION
 Recombinant proteins which have
histidine tags can be purified using
immobilized metal ion chromatography
(IMAC).
 The His-tag can be placed on either the
N- or C-terminus.
 Optimal binding and, therefore,
purification efficiency is achieved when
the His-tag is freely accessible to metal
ion support.
4. BIOTIN & BIOTINYLATED
MOLECULES PURIFICATION
• If a biotin tag can be
incorporated into a
biomolecule, it can be used
to purify the biomolecule
using a streptavidin or avidin
affinity support.

32. 6.AFFINITY PURIFICATION
OF ALBUMIN
&MACROGLOBULIN
CONTAMINATION
 Affinity purification is a helpful tool for
cleaning up and removing excess albumin
and α2- macroglobulin contamination
from samples since these components can
mask or interfere with subsequent steps of
analysis
5. ENZYME ISOLATION
• There are a number of areas related to
affinity chromatography that have also
been of great interest in
pharmaceutical and biomedical
analysis.
• One such area is in the use of affinity
columns for enzyme isolation.
• This approach may use affinity ligands
that are substrates, inhibitors,
cofactors, or proteins that are
associated with the biochemical
pathways of the target enzyme

33. 7. PROTEIN AND PEPTIDE SEPARATIONS
 Affinity chromatography has a wide range of applications for protein
purification, such as immunoaffinity, purification of immunoglobulins,
purification of glycoproteins, DNA-binding proteins, receptor proteins,
enzymes, cell isolation, and nucleotide isolation.
 Affinity chromatography is a specific purification technology based on
biological function or individual chemical structure.
 The application of this technique is in separation of active biomolecules
from denaturated or functionally different forms in the isolation of pure
proteins present at low concentration and also for removing specific
contaminants.

34. 8. INVESTIGATING PROTEIN-PROTEIN INTERACTIONS
 Affinity chromatography provides one important method for identifying
and characterizing the intermolecular interactions.
 When investigating protein-protein interactions, affinity
chromatography typically involves linking one protein to an insoluble
matrix and then incubating that matrix with a solution containing
possible binding partners.
 This solution could be as simple as homogenous solution containing a
single recombinant protein or complex.
 After incubating the immobilized substrate protein with its potential
binding partner(s) and washing away material associated non
specifically the binding partner are the eluted and detected by any
variety of methods from chromatographic detection

35.  Extremely high-specificity
 The purest of levels can be achieved
 The process is highly reproducible.
 The binding sites of biological molecules could be investigated by simply
looking at the binding sites of biological molecules.
 Single-step purification.
 The matrix is reusable in a short time and is solid that is easy to clean and
dried.
 Provide purified products with high yield.
 Affinity chromatography may also be used to get rid of particular

36.  Expensive ligands
 Leakage of the ligand
 Degradation of the solid support
 Relatively low productivity
 Non-specific adsorption can not be totally eliminated, it can only be
minimized.
 The limited life span and the high cost for immobilized ligands.
 Proteins are denatured when the necessary pH is not maintained.