Affinity chromatography is a sample purification technique,used primarily for biological molecules such as proteins. 1.Principle 2.Theory 3.Instrumentation 4. Applications

1. AFFINITY
CHROMATOGRAPHY
PreparedBY:
REKHA T N
M Pharma
DEPT. OF PHARMACEUTICALANALYSIS
SACCP
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2. CONTENT
• History
• Introduction
• Principle
• Theory
• Procedure
• Application
• Advantages
• Disadvantages
• Reference
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3. AFFINITY HISTORY
• 1930s, first developed by A. Wilhelm Tiselius-a Swedish
biochemist, won the Nobel prize in 1948.
• Used to study enzymes and other proteins.
• Relies on the affinity of various biochemical compounds with
specific properties.
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4. INTRODUCTION
• Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases i.e.,
stationary phase and mobile phase which moves in a definite
direction.
• Affinity chromatography is 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 ligand, which is immobilized on a support.
• If a solution of a mixture of proteins is passed through the
chromatographic column one of the proteins binds to the ligand based
on the specificity an high affinity (they fit together like a lock and
key).
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5. • The other proteins in the solution wash through the column
because they were not able to bind to the ligand.
ligand
bead
target proteins
Free
ligand
Elute the bound target proteins
by introducing the free ligand
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6. PRINCIPLE
• Affinity chromatography is one of the most diverse and
powerful chromatographic methods for purification of a
specific molecule or a group of molecules from complex
mixtures.
• It is based on highly specific biological interactions between
two molecules such as interactions between enzyme and
substrate, receptor and ligand, or antibody and antigen.
• These interactions which are typically reversible are used for
purification by placing one of the interacting molecules
referred to as affinity ligand onto a solid matrix, to create a
stationary phase while a target molecule is in the mobile phase.
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8. • The sample is injected into the equilibrated affinity
chromatography column.
• Only the substance with affinity for the ligand are retained on
the column.
• The substance with no affinity to the ligand will elute off.
• The substances retained in the column can be eluted off by
changing the pH of salt or organic solvent concentration of the
elute.
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9. THEORY
• Specificity of affinity chromatography:
Specificity is based on three aspects of affinity
a. Matrix
b. Spacer arm
c. Ligand.
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10. MATRIX
• The matrix simply provides a structure to increase the surface area
to which the molecule can bind.
• Amino, hydroxyl and carbonyl groups located with the matrix
serve as ligand binding sites.
• Matrix are made up of agarose and other polysaccharides.
For having an effective matrix, it must have a certain characters:
 It must be chemically and mechanically stable.
 It must be insoluble in solvents and buffers employed in the
process.
 It must be easily coupled to a ligand or spacer arm onto which
the ligand can be attached.
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11.  It must exhibit good flow properties and have a relatively
large surface area for attachment.
Types of matrix used:
Cellulose: used for DNA affinity chromatography
Agarose: DNA or proteins
Tris-acryl:
 It is having higher separation ability
 It has small particle size 40-80 micrometer.
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12. SPACER ARM
• The stationary phase is typically a gel matrix, to prevent steric
interference or overlap during the binding process of the target
molecule to the ligand, an inhibitor containing a hydrocarbon
chain is first attached to the agarose bead (solid support).
• This inhibitor with a hydrocarbon chain is commonly known
as the spacer between the agarose bead and the target
molecule.
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13. LIGAND
• The ligand binds only to the desired molecule within the
solution.
• It attaches to the matrix which is made up of an inert
substance.
• It should only interact with the desired molecule and form a
temporary bond.
• The ligand/complex molecule will remain in the column,
eluting everything else off.
• The ligand/molecule complex dissociates by changing the pH.
• The ligand can be selected only after the nature of the
macromolecule to be isolated is known.
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14. • The chosen ligand must bind strongly to the molecule of
interest
• If the ligand can bind to more than one molecule in the sample
a Negative affinity is performed.
– This is the removal of all ligands, leaving the molecule of
interest in the column.
– Done by adding different ligands to bind to the ligands
within the column.
• The selection of the ligand for affinity chromatography is
influenced by two factors:
– The ligand must exhibit specific and reversible binding
affinity for the target substances.
– It must have chemically modifiable groups that allow it to
be attached to the matrix without destroying binding
activity.
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15. • Immobilization of ligands:
– Immobilization of affinity ligand is very important when
designing an affinity chromatography method for
biomolecule purification.
– Activity of the affinity ligand can be affected by multi-site
attachment, multi-site attachment occurs when an affinity
ligand is attached through more than one functional group
on a single ligand molecule.
– These multiple attachment sites causes the ligand to
become denatured or distorted, multi site attachment can
lead to reduced binding affinity.
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16. • Covalent immobilization:
– Covalent immobilization is one of the most common ways
of attaching an affinity ligand molecule to a solid support
material.
– There are a wide range of coupling chemistries available
when considering covalent immobilization methods.
– Amine, sulfhydryl, hydroxyl, aldehyde, and carboxyl
groups have been used to link affinity ligands.
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17. • Adsorption of affinity ligands;
– Adsorption can be either nonspecific or specific.
• Non specific adsorption: in Non specific adsorption the
affinity ligand simply adsorbs to the surface of the
support material and is a result of hydrogen bonding
and/or hydrophobic interactions.
• Bio-specific adsorption: it is commonly performed by
using avidin or streptavidin for the adsorption of biotin
containing affinity ligands for the adsorption of
antibodies in and/or hydrophobic interactions.
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18. Attachment of ligand to matrix.
 Several procedures have been developed for the covalent
attachment of the ligand to the stationary phase.
 All procedures for gel modifications proceed in two
separate chemical steps:
a. Activation of the functional group on the matrix.
b. Joining of the ligand to the functional group on the
matrix.
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19. • Examples:
o Antigen Antibody
o Antibody Antigen
o Substrate Enzyme
o DNA Histon
o Hormone Binding protein/receptor.
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20. Procedure
• Affinity chromatography technique involves three main steps:
– Bind
– Wash
– elute
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21. • Step-1: Attach ligand to column matrix.
– Binding of the selected ligand to column matrix requires a
covalent bond to be formed between them.
– Most ligands are attached first to spacer arms and then
bonded to matrix. The ligand matrix gel is then loaded into
an elution column.
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22. – Once the column has been prepared,
the mixture containing isolate is
poured into the elution column.
– Gravity pulls the solution through the
gel, because most of the proteins do
not bind to the ligand matrix complex.
– When ligand is 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 its gravity, but most
remain unbound in the gel column.
• Step-2: Load protein mixture onto column.
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23. • Step-3: Proteins bind to ligand.
– 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.
– Once the impurities are washed
out, the only remaining part of the
protein mixture should be the
desired isolates.
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24. • Step-4: Wash column to remove unwanted
material
– In ally to collect isolate, which is still bound to the ligand
matrix in the gel, a stronger second wash is run through the
column.
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25. • Step-5: Wash of proteins that bind loosely
– 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.
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26. • Step-6: Elute proteins that bind tightly to ligand
and collect purified protein of interest.
– The protein is then free to run through the gel and be
collected.
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27. Application
• Immunoglobulin purification (antibody immobilization)
– Used to purify antibody against a specific antigen
Ex: Immunoglobulin's
– 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’- carbonyl di-imidazole,
tresylchloride or tosyl chloride.
– As these are random immobilization methods, the antibody binding
sites may be blocked due to improper orientation, multi-site
attachment or steric hindrance.
– Antibodies can also be immobilized by adsorbing them onto
secondary ligands.
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28. • 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.
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29. • GST tagged purification
– The purification method is based on the high affinity of GST
for glutathione.
– When applied to the affinity resin, GST-tagged proteins bind
to the glutathione ligand, and impurities are removed by
washing with binding buffer.
– Tagged proteins are then eluted from the chromatography
resin under mild, non-denaturing conditions that preserve
both protein structure and function.
– GST Buffer Kit contains prepared buffer concentrates for
binding, washing, and elution of GST tagged protein
detection.
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30. • His-tagged protein purification
– Histidine-tagged recombinant protein purification using
Immobilized metal ion chromatography(IMAC).
– Ni2+ Sepharose resins are precharged with nickel ions
(Ni2+) metal ion affinity chromatography.
– Ni2+ Sepharose excel is especially suitable for purification of
histidine tagged proteins secreted into eukaryotic cell culture
supernatants.
– IMAC resins charged with Ni2+ and Co2+ are the most
commonly used methods for the purification of histidine
tagged proteins.
– However, in some cases, other metal ions may be more
suitable, for example copper ( Cu2+) or zinc (Zn2+). In these
cases, uncharged IMAC resins can be conveniently charged
with the metal ion of your choice.
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31. • Protein A, G, and L purification:
– Proteins A, G, and L are native or recombinant proteins of
microbial origin which bind specifically to
immunoglobulins including immunoglobulin G (IgG).
– The most popular matrixes or supports for affinity
applications which utilize protein A, G, or L is beaded
agarose (e.g. Sepharose CL-4B; agarose cross-linked with
2,3dibromopropanol and desulphated by alkaline
hydrolysis under reductive conditions), polyacrylamide,
and magnetic beads.
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32. • Biotin and biotinylated molecules purification:
– Biotin:(vitamin H or B7) cofactor in the metabolism of
fatty acids and leucine, and in gluconeogenesis
– In affinity chromatography it is often used an affinity tag
due to its very strong interactions with avidin and
streptavidin. One advantage of using biotin as an affinity
tag is that it has a minimal effect on the activity of a large
biomolecule due to its small size (244 Da).
– Streptavidin is a large protein (60 kDa) that can be obtained
from Streptomyces avidinii and bind biotin. Avidin is a
slightly larger glycoprotein (66 kDa) with slightly stronger
binding to biotin . Both avidin and streptavidin have four
subunits that can each bind one biotin molecule.
– Due to the strong interaction between biotin and
(strept)avidin, harsh elution conditions are required to
disrupt the binding.
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33. • Lectin affinity chromatography:
– Lectins are carbohydrate binding proteins that contain two or
more carbohydrate binding sites and can be classified into five
groups according to their specificity to the monosaccharide.
– They exhibit the highest affinity for: mannose, galactose/N
acetyl galactosamine, N acetyl glucosamine, fructose, and N-
acetyl neuraminic acid.
– In this affinity technique, protein is bound to an immobilized
lectin through its sugar moieties .
– Once the glycosylated protein is bound to the affinity support,
the unbound contaminants are washed away, and the purified
protein is eluted.
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34. • 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.
– 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.
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35. • Reversed phase chromatography
– Reversed phase chromatography is a kind of affinity
interaction between a biomolecule dissolved in a solvent
(mobile phase) that has some hydrophobicity (e.g. proteins,
peptides, and nucleic acids) and an immobilized
hydrophobic ligand (stationary phase).
– When using reversed phase chromatography, the most polar
macromolecules are eluted first and the most nonpolar
macromolecules are eluted last: the more polar
(hydrophilic) a solute is, the faster the elution and vice
versa. table for separating non-volatile molecules.
– Initial step of reversed phase separation involves
equilibration of the column under suitable conditions (pH,
ionic strength and polarity.
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36. – Next, sample is applied and bound to the immobilized
matrix.
– Following this step, desorption and elution of the
biomolecules is achieved by decreasing the polarity of the
mobile phase (by increasing the percentage of organic
modifier in the mobile phase).
– At the end of the separation, the mobile phase should be
nearly 100% organic to ensure complete removal of all
bound substances.
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37. Advantages
• Extremely high specificity
• High degree of purity can be obtained
• The process is very reproducible
• The binding sites of biological molecules can be
simply investigated.
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38. Disadvantages
• Expensive ligands
• Leakage of ligand
• Degradation of the solid support
• Limited lifetime
• Non-specific adsorption
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39. • References
– Cuatrecasas P. Protein purification by affinity
chromatography: derivatizations of agarose and
polyacrylamide beads. Journal of Biological Chemistry.
1970 Jun 25;245(12):3059-65.
– Uhlén M. Affinity as a tool in life science. Biotechniques.
2008 Apr;44(5):649-54.
– Hage DS. Affinity chromatography: a review of clinical
applications. Clinical chemistry. 1999 May 1;45(5):593-615.
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