Chromatography

1. AFFINITY
CHROMATOGRAPHY
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2. HISTORY
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 1930s, first developed by Arne Wilhelm Tiselius, won the
Nobel Prize in 1948
 Used to study enzymes and other proteins
 Relies on the affinity of various biochemical compounds
with specific properties
ex) enzymes for their substrates
antibodies for their antigens

3. 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).
 The other proteins in the solution wash through the column
because they were not able to bind to the ligand.
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4. PRINCIPLE
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 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.
 Many of the commonly used ligands coupled to affinity
matrices are now commercially available and are ready
to use.

5. EXAMPLES
• Antigen
• Substrate
Antibody
Enzyme
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6. 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 eluent
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7. Specificity of Affinity Chromatography
 Specificity is based on three aspect of affinity
Matrix: for ligand ttachment.
Matrix should be chemically and
physically inert.
Spacer arm: used to improve
binding between ligand and
target molecule by overcoming
any effects of steric hindrance.
Ligand: molecule that binds
reversibly to a specific target
molecule(site of interaction).
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8. Matrix (solid support)
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 The matrix simply provides a basic
structure to increase the surface area to
which the molecule can bind.
 The matrix must be activated for the
ligand to bind to it but still able to retain
it’s own activation towards the target
molecule.

9. Matrix
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 Amino, hydroxyl, carbonyl and thio groups located with
the matrix serve as ligand binding sites
 Matrix are made up of agarose and other
polysaccharides
 The matrix also must be able to withstand the
decontamination process of rinsing with sodium
hydroxide or urea

10.  Solid support:
a. carbohydrate polymers: ex, agarose, cellulose, dextran.
Commercially available agarose in 1-12% is usually
adequate.
cross linking with divinylsulfone(DVS) or 1,4-butanediol
diglycidyl ether increases both the chemical and thermal
stability of the gel but may diminish the binding capacity.
DVS increases the gel’s rigidity.
Cross linked agarose tolerates any solvents except strong
acids and oxidizers but may be damaged by some rare
enzymes and must not be frozen or dried. Agarose gel
activates complement system in human blood and must
never be used in vivo ( e.g., for affinity removing of toxins
from blood).
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11.  Dextran: it consists of two types of gels a. sephadex b.
sephacryl
 Sephacryl are used in the chromatography of
biomolecules. Sephacryl S-1000 is especially suitable for
affinity chromatography of very large molecules. At low
pH Sephacryl S-200 adsorbs proteins.
 Sephadex is used mainly as a glucose polymer
(example for the purification of lectins ) it is
mechanically to weak or has insufficient porosity . It can
be activated in aqueous media with non-cross-linking
reagents.
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12.  B. Synthetic polymers:
 polyacrylamide(PAA) is synthesized by copolymerization of
acrylamide and a cross linking agent. Primary disadvantage
is that the gels are either soft or have small pores. PAA gel
is resistant against enzymatic attacks and does not adsorb
biomolecules.
 Hydroxyalkylmethacrylate gels: the gel is chemically and
mechanically more stable than PAA gel but is more
hydrophobic and thus it is inappropriate for many
applications.
 Trisacryl: it is hydrophilic, biologically inert, rigid, and
macroporous. Despite its low working volume( it contains
more than 30% of solids) it is used for large scale
preparations.
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13.  C. mixed gels:
Polyacrylamide-agarose gels (ultrogel AcA) were
developed for gel filtration and tested for affinity
chromatography.
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14.  D. inorganic materials:
When extreme rigidity of the support is needed , as
for HPLC and large-scale applications, inorganic materials
are used ( irregular porous glass or silica particles). They
are soluble pH >8, although a coating of zirconium will
enhance their stability. otherwise, glass or silica may be
coated with an inert polymer.
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15.  E. magnetic carriers :
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Magnetic sorbent particles (magnogel AcA 44 and Act-
magnogel AcA 44) are useful for batch extraction from
large volumes of diluted and/or turbid solutions.

16. Ligand
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 The Ligand binds only to the desired molecule within the solution
 The ligand attaches to the matrix which is made up of an inert
substance
 The ligand should only interact with the desired molecule and form a
temporary bond
 The ligand/molecule complex will remain in the column, eluting
everything else off
 The ligand/molecule complex dissociates by changing the pH

17. ligand
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 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 technique, 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.

18.  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 substance(s) and
it must have chemically modifiable groups that allow
it to be attached to the matrix without destroying binding
activity.
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19. Immobilization of ligand
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 Immobilization of the affinity ligand is also 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. orientation of the affinity ligand,
and If these multiple attachment sites cause the affinity
ligand to become denatured or distorted, multisite
attachment can lead to reduced binding affinity.
However, in some instances, the additional attachment
sites can result in more stable ligand attachment steric
hindrance.

20. Covalent immobilization
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 Covalent immobilization is one of the most common
ways of attaching an affinity ligand to a solid support
material. There is 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.
 covalent attachment methods are more selective than
other immobilization methods, they generally require
more steps and chemical reagents onto support
materials.

21. Adsorption of affinity ligands
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 adsorption can be either nonspecific or specific.
Nonspecific adsorption: In nonspecific adsorption the
affinity ligand simply adsorbs to the surface of the support
material and is a result of Coulombic interactions, 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 or protein A or protein G for the
adsorption of antibodies in, and/or hydrophobic
interactions.

22. Space arm
 the binding sites of the target
molecule are sometimes
deeply located and difficult to
access due to steric
hindrance, a spacer arm is
often incorporated between
the matrix and ligand to
facilitate efficient binding and
create a more effective and
better binding environment.
The length of these spacer arms
is critical. Too short or too long
arms may lead to failure of
binding or even non-specific
binding. In general, the spacer
arms are used when coupling
molecules less than 1000 Da.
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23.  Affinity purification involves 4 steps:
1. preparing the sample to load on the affinity column.
2. loading and incubation of the sample with ligand to
promote binding
3.washing away the non bound components from the
column
4. eluting(recovering) the bound components.
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25. Elution
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 Adsorbed compounds can be washed off the column in two
ways: by specific ( concurrent) or by non specific elution.
 Specific elution is based on direct interruption by analogs of
either ligand or adsorbate in to the complex formed on the
affinity resin.
 non specific elution is by changing the media atmosphere
(e.g. changing the ionic strength, pH or polarity)
 If the affinity interaction is mainly hydrophobic, elution with
detergent or mixed solvents( up to 50% ethylene glycol or
isopropanol) is promising but if the interaction is ionic, high
salt concentration or pH alteration are used.
 Elution from antigen-antibody complexes can be modeled
on polystyrene microplates: immune complexes are
dissociated without eluting the adsorbed antigen, while all
parameters are controlled by ELISA technique.

26.  pH elution:
A change in pH alters the degree of ionization of charged
groups on the ligand and/or the bound protein. A step
decrease in pH is the most common way to elute bound
substances. The chemical stability of the matrix, ligand and
target protein determines the limit of pH that may be used
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27.  Ionic strength elution:
The exact mechanism for elution by changes in ionic
strength will depend upon the specific interaction between
the ligand and target protein. This is a mild elution using a
buffer with increased ionic strength (usually NaCl), applied
as a line.
e.g., Enzymes usually elute at a concentration of 1 M NaCl
or lesser gradient or in steps.
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28.  Competitive elution: Selective eluents are often used to
separate substances on a group specific medium or
when the binding affinity of the ligand/target protein
interaction is relatively high. The eluting agent competes
either for binding to the target protein or for binding to
the ligand. Substances may be eluted either by a
concentration gradient of a single eluent.
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29. . Applications of affinity
chromatography
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 1 Immunoglobulin purification (antibody
immobilization) :
 Used to purify antibody against a specific antigen
Ex:Immunoglobulins
 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. 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.

30. Recombinant tagged proteins :
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 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.
 most commonly used tags are glutathione-S-transferase
(GST), histidine fusion (His or poly His tag) and protein
A fusion tags. Other types of fusion tags are also
available including maltose-binding protein , thioredoxin,
NusA ,GB1 domain for protein G

31. 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,
31washing, and elution of GST-
tagged protein detection

32. His-tagged protein purification
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 histidine-tagged recombinant protein purification using
immobilized(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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34. Protein A, G, and L purification:
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 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.

35. Biotin and biotinylated molecules
purification:
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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).

36.  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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37. Lectin affinity chromatography:
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 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/Nacetylgalactosamine, N-
acetylglucosamine, fructose, and N-acetylneuraminic
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.

38. Nucleic acid separation using immobilized
metal affinity chromatography (IMAC)
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 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

39.  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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40. Reversed phase chromatography
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 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.

41.  initial step of reversed phase separation involves
equilibration of the column under suitable conditions
(pH, ionic strength and polarity.
 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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42.  Reference:
 Affinity chromatography by GE healthcare
(http://proteins.gelifesciences.com/~/media/protein-
purification-ib/documents/handbooks/affinity-
chromatography-handbook-vol-2.pdf)
 Affinity chromatography: principles and applications-
InTech open
(http://cdn.intechopen.com/pdfs/33046.pdf)
 Affinity chromatography - journal homepage:
www.elsevier.com/locate/ymeth.
 https://www.slideshare.net/imaginarybiologist/affinity-
chromatography
 www.google.com (images)
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