AFFINITY CHROMATOGRAPHY

1. PRESENTATION
ON
ROPRINCIPLE AND APPLICATIONS OF AFFINITY CHMATOGRAPHY
Presented by: Guided by:
Mohd Fahad Mr. Pramdeep Bagga
M. Pharma Ist Year Asstt. Professor
(Pharmacology) Faculty of Pharmacy
INTEGRAL UNIVERSITY LUCKNOW
FACULTY OF P[HARMACY

2. INTRODUCTION
• Affinity Chromatography is essentially a sample purification
technique, used primarily for biological molecules such as
proteins.
• It is a method of separating a mixture of proteins or nucleic
acids (molecules) by specific interactions of those molecules
with a component known as a ligand, which is immobilized on
a support. If a solution of, say, a mixture of proteins is passed
over (through) the column, one of the proteins binds to the
ligand on the basis of specificity and high affinity (they fit
together like a lock and key).

3. Principle:
•The stationary phase is typically a gel matrix, often of agarose; a linear
sugar molecule derived from algae.
•Usually the starting point is an undefined heterogeneous group of
molecules in solution, such as a cell lysis, growth medium or blood
serum.
•The molecule of interest will have a well known and defined property,
and can be exploited during the affinity purification process.
•The process itself can be thought of as an entrapment, with the target
molecule becoming trapped on a solid or stationary phase or medium.

4. •The stationary phase can then be removed from the mixture,
washed and the target molecule released from the entrapment in a
process known as elution.The other molecules in the mobile phase
will not become trapped as they do not possess this property.
•Possibly the most common use of affinity chromatography is for
the purification of recombinant proteins

5. Column step

6. Batch steps

7. A third method, expanded bed adsorption, which combines the
advantages of the two methods mentioned above, has also been
developed. The solid phase particles are placed in a column where
liquid phase is pumped in from the bottom and exits at the top. The
gravity of the particles ensure that the solid phase does not exit the
column with the liquid phase.
Affinity columns can be eluted by changing salt concentrations, pH, ,
charge and ionic strength directly or through a gradient to resolve
the particles of interest.

9. Various affinity media
• Many different affinity media exist for a variety of possible
uses
• Amino Acid- Used with a variety of serum proteins, proteins,
peptides, and enzymes, as well as rRNA and dsDNA
• Avidin Biotin – Used in the purification process of
biotin/avidin and their derivatives.
• Carbohydrate Bonding – Most often used with glycoproteins
or any other carbohydrate-containing substance.
• Carbohydrate – Used with lectins, glycoproteins, or any other
carbohydrate metabolite protein.

10. •Dye Ligand – This media is nonspecific, but mimics biological substrates
and proteins.
•Glutathione – Useful for separation of GST tagged recombinant
proteins.
•Heparin – This media is a generalized affinity ligand, and it is most
useful for separation of plasma coagulation proteins, along with nucleic
acid enzymes and lipases.
•Hydrophobic Interaction – Most commonly used to target free carboxyl
groups and proteins.
•Immunoaffinity – Detailed below, this method utilizes antigens' and
antibodies' high specificity to separate.

11. •Immobilized Metal Affinity Chromatography – Detailed further below,
this method uses interations between metal ions and proteins (usually
specially tagged) to separate.
•Nucleotide/Coenzyme – Works to separate dehydrogenases, kinases,
and transaminases.
•Nucleic Acid – Functions to trap mRNA, DNA, rRNA, and other nucleic
acids/oligonucleotides.
•Protein A/G – This method is used to purify immunoglobulins.
•Specialty – Designed for a specific class or type of protein/coenzyme,
this type of media will only work to separate a specific protein or
coenzyme.

12. • Thus, double-stranded plasmid and genomic DNA, which
has no low binding affinity can be easily separated from
RNA or oligonucleotides which bind strongly to metal-
charged chelating matrices
• IMAC columns clarify plasmid DNA from bacterial
alkaline lysates, purify a ribozyme, and remove
primers and other contaminants from PCR
reactions

13. Applications
• Used in Genetic Engineering
- nucleic acid purification
• Production of Vaccines
- antibody purification from blood
serum
• And Basic Metabolic Research
- protein or enzyme purification from
cell free extracts

14. biotinylation
means affinity chromatography using
immobilized avidin or streptavidin
to separate the biotinylated protein from a
mixture of other proteins and biochemicals

15.  Avidin(or strepdavidin) -biotin interaction is used to purify
proteins
Avidin:protein deposited in the whites of eggs(birds,reptiles…)
Streptavidin is a tetrameric protein purified from the bacterium
Streptomyces avidinii
Biotin:(vitamin H or B7) cofactor in the metabolism of fatty acids
and leucine, and in gluconeogenesis
PROTEIN PURIFICATION

16.  The non-covalent bond formed between biotin and avidin or
streptavidin
 has a binding affinity >most antigen and antibody bonds
 ~strength of a covalent bond

17. Nucleic acid separation using immobilized metal
affinity chromatography (IMAC)
• The method can be used to purify compounds
containing purine or pyrimidine moieties where
the purine and pyrimidine moieties are shielded
from interaction with the column matrix from
compounds containing a non-shielded purine or
pyrimidine moiety or group.

18. ADVANTAGES OF AFFINITY
CHROMATOGRAPHY
1) Extremely high specificity
2) High degrees of purity can be obtained
3) The process is very reproducible
4) The binding sites of biological molecules can
be simply investigated

19. DISADVANTAGES OF AFFINITY
CHROMATOGRAPHY
1) Expensive ligands
2) Leakage of ligand
3) Degradation of the solid support
4) Limited lifetime
5) Non-specific adsorption
6) Relatively low productivity

20. THANK YOU