Affinity Chromatography

2. Presented by:
Samya Sayantan
Id: 121-29-381
Batch: 7th
Sec: A
Department of Pharmacy
Daffodil International University

3. Introduction:
Affinity chromatography is principally based on the molecular
recognition of a target molecule by a molecule bound to
column.
Affinity purification involves 3 main steps:
a. Incubation of a crude sample with the affinity support to
allow the target molecule in the sample to bind to the
immobilized ligand.
b. Washing away non-bound sample components from the
support.
c. Elution (dissociation and recovery) of the target molecule
from the immobilized ligand by altering the buffer conditions
so that the binding interaction no longer occurs.

4. Definition:
Affinity Chromatography is a separation method based on a
specific binding interaction between an immobilized ligand
and it’s binding partner.
Example:
Antigen Antibody
Antibody Antigen
Substrate Enzyme
DNA Histon
Hormone Binding Protein/Receptor

5. Principle:
 The stationary phase is typically as gel matrix (often
agarose).
 The molecule of interest has a known and defined property.
 The process is an entrapment in which the target molecules
becomes trapped on stationary phase.
 The stationary phase can then be removed from the
mixture, washed and then target molecules is released from
the entrapment.

7. Chromatographic Media
A matrix in its use here is a substance, usually in bead
form to which a specific ligand is covalently bound.
In order to for the matrix to be effective it must have
certain characters:
 It must be insoluble in solvents and buffers employed
in the process.
 It must be chemically and mechanically stable.
 It must be easily coupled to a ligand or spacer arm
onto which the ligand can be attached.
 It must exhibit good flow properties and have a
relatively large surface area for attachment

8. Immobilized Ligand:
 The ligand can be selected only after the nature of the
macromolecule to be isolated is known.
 When a hormone receptor protein is to be purified by affinity
chromatography, the hormone itself is an ideal candidate for
the ligand.
 For antibody isolation, an antigen or hapten may be used as
ligand.
 If an enzyme is to be purified, a substrate analog, inhibitor,
cofactor, or effectors may be used as a the immobilized
ligand.

9. Attachment of Ligand:
Several procedures have been developed for the covalent
attachment of the ligand to the stationary phase.all
procedures for gel modification proceed in two separate
chemical steps:
 Activation of the functional groups on the matrix and
 Joining of the ligand to the functional group on the matrix.

10. Materials:
 A bead matrix.
 A ligand.
 A solution containing the substrate to be isolated a wash to
elute the non-bound impurities in the solution.
 A final wash to elute the bound substrate from its ligand.
The bead matrix is an agarose gel loaded into an elution
column. Sepharose is the most widely used matrix, because
the hydroxyl groups on the sugar residues can be easily
manipulated to accept a ligand.

11. Procedure:
 Binding of the selected ligand to the matrix requires that a
covalent bond be formed between the two. Most ligands are
attached first to spacer arms which are then bonded to the
matrix. The ligand-matrix gel is then loaded into an elution
column.
 Once the column has been prepared, the mixture containing
your favorite isolate is poured into the elution column. Once
in the column, gravity pulls the solution through the gel,
because most of the proteins do not bind to the ligand-matrix
complex. However, when the ligand's recognized
substrate passes through the gel, it binds to the ligand-matrix
complex, halting its passage through the gel. Some of
the impurities flow through the gel due to gravity, but most
remain, unbound, in the gel column.

12.  In order to remove these unbound impurities, a wash of
extreme pH, salt concentration, or temperature is run
through the gel. It is important to use a strong wash so that
all the impurities are removed, but it is also just as crucial
that the wash be not so strong that it removes the bound
isolates. Once the impurities are washed-out, the only
remaining part of the protein mixture should be the
desired isolates.
 Finally to collect the isolate which is still bound to the
ligand-matrix in the gel, a stronger second wash is run
through the column. This second wash relies on the
reversible binding properties of the ligand which allows
the bound protein to dissociate from its ligand in the
presence of this stronger wash. The protein is then free to
run through the gel and be collected.

14. Application:
 Purifying a protein based on histidine –nickel
interaction:
A major problem in the field of molecular biology involves the
expression and purification of a protein of interest, for
example the human adenovirus serotype, Ad5. This protein
has been commonly used as a vector for gene therapy, due
to its efficient gene delivery and application in a diverse
range of cells. A simple one step metal affinity purification
system, utilizing the His tag which binds to Ni-NTA, was
used to isolate the Ad5, with greater than 95% purity. The
isolated protein was evaluated via a receptor-binding assay
in Hela cells.

16.  Purifying a protein based on inherent ligant-binding
specificity:
There are a few proteins that have natural affinity to bind metal
ions. This inherent metal ion binding has been utilized as a way
to purify these proteins using metal immobilized affinity
columns. For example, a red fluorescent protein, DsRed, has a
binding affinity for copper ions. On the basis of this a
purification method for DsRed was developed using copper
immobilized column. A metal chelating ligand attached to beads
was used in this study to immobilize copper ions on beads. The
crude protein was passed through the copper immobilized
beads. DsRed bound to the beads whereas other interfering
proteins did not bind to the beads and hence were removed.
DsRed was eluted using a competitive ligand imidazole that
binds to copper ions. This is a simpler approach of protein
purification based on their inherent metal ions affinity.

17.  Study of drug interaction:
The quantitative characterization of interactions between
targets and ligands is an integral part of the drug discovery
process. These interactions are often defined by the
association constant of a drug-protein interaction. The
association constants, for a number of HSA-binding drugs
based upon their retention factors. They prepared an
immobilized HSA column by passing known
concentrations of the drugs through the column saturation
plots were generated which were used to calculate
retention factors and association constants. This system
offers advantages over the more common methods of
evaluating association constants such as fast response times
ease of automation, and additionally the ability to
distinguish chiral compounds simultaneously.

18. Advantages
 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

19. Disadvantages
 Expensive ligands
 Leakage of ligand
 Degradation of the solid support
 Limited lifetime
 Non-specific adsorption
 Relatively low productivity