Introduction to chromatography, history, Principle, a brief classification, affinity chromatography in detail, Principle, common terms like matrix, spacer arm, ligand classification in detail, ligand immobilisation, entrapment, sample preparation, elution and its types, Processing steps, applications of affinity chromatography in detail, Troubleshooting Problems of affinity chromatography based on elutants and remedies graphically in detail.

1. INTRODUCTION
CHROMATOGRAPHY
Prepared By,
Ranjith. R
M. Pharma – 1st-Sem
Krupanidhi college of Pharmacy
1

2. CHROMATOGRAPHY
2

3. BEFORE CHROMATOGRAPHY
3

4. EXTRACTION
• Extraction is a way to separate a desired substance
when it is mixed with others.
• The mixture is brought into contact with a solvent in
which the substance of interest is soluble, but the other
substances present are insoluble.
• Extractions use two immiscible phases (these are
phases that do not mix, like oil and water) to separate
the substance from one phase into the other.
Example of Extraction
Tea-making is a very basic non-laboratory
extraction. You boil tea leaves in water to extract the
tannins, theobromine, polyphenols, and caffeine out of
the solid tea leaves and into the liquid water. You are then
free to drink and enjoy the water and the extracted
substances it contains.
4

5. CRYSTALLIZATION
Crystallization is the solidification of atoms or
molecules into a highly structured form called a
crystal.
Crystallization can also refer to the solid-liquid
separation and purification technique in which mass
transfer occurs from the liquid solution to a pure solid
crystalline phase.
5

6. • DISTILLATION
Distillation is the technique of heating a liquid to
create vapour which is collected when cooled
separately from the original liquid.
It's based on the different boiling point or volatility
values of the components. The technique may be
used to separate components of a mixture or to aid in
purification.
6

7. • FILTRATION
Filtration is a process used to
separate solids from liquids or gases using a filter
medium that allows the fluid to pass, but not the
solid.
The term "filtration" applies whether the filter is
mechanical, biological, or physical. The fluid that
passes through the filter is called the filtrate.
The filter medium may be a surface filter, which is a
solid that traps solid particles, or a depth filter,
which is a bed of material that traps the solid.
7

8. Just like the previous techniques chromatography is a
way to separate two or more components based on
specific characteristics.
The results are reproducible with better accuracy than
the before mentioned separation techniques.
Chromatography can separate more complex mixtures
than the previous techniques.
Chromatography is less time consuming and cheaper.
Then why chromatography is more preferable?
8

9. Brief History of chromatography
1903 – Tswett, a Russian botanist coined the term chromatography. He passed plant
tissue extracts through a chalk column to separate pigments by differential adsorption
chromatography.
1915 – R.M Willstatter, German Chemist won Nobel Prize for similar experiment.
1922 – L.S Palmer, American scientist used Tswett’s techniques on various natural
products.
1931 – Richard Kuhn used chromatography to separate isomers on polyene pigments;
this is the first known acceptance of chromatographic methods.
1938 – Thin Layer chromatography by Russian scientist N.A Izamailov and M.S Shraiber.
1941 – Liquid –Liquid partition chromatography developed by Archer John, Porter
Martin and Richard Laurence Millington Synge.
1944 – Paper Chromatography analytical gas-solid (adsorption) chromatography
developed by Fritz Prior.
9

10. 1950 – Gas Liquid Chromatography by Martin and
Anthony James; Martin won the Nobel Prize in 1952.
1966 – HPLC named by Csaba Hovarth, but didn’t
become a popular method until 1970’s.
1970’s – Ion-exchange Chromatography was
developed by Hamish Small and co-workers at the
Dow Chemical company.
1930’s – Affinity Chromatography was developed for
the study of enzymes and other proteins.
10

11. Chromatography definition
Chromatography is a powerful separation
method that finds application to all branches of
science.
Chroma means “color” and graphein means to
“write”.
• What is Chromatography?
A method of separating and analysing mixtures
of chemicals.
The separation, especially of closely related
compounds, by allowing a solution or mixture to seep
through an adsorbent (such as clay, gel, or paper) so
each compound becomes adsorbed into a separate,
often coloured, layer. 11

12. PRINCIPLE:
In all chromatographic separations the sample is
transferred in a mobile phase, which may be a gas , a
liquid, or a supercritical fluid.
This mobile phase is then forced through an immiscible
stationary phase, which is fixed in place in a column or on
a solid surface.
The two phases are chosen so that the components of
the sample distribute themselves between the mobile
and stationary phase to varying degrees.
Those components that are strongly retained by the
stationary phase move only slowly with the flow of
mobile phase.
As a consequence of these differences in solubility,
sample components separate into discrete bands, or
zones, that can be analyzed qualitatively and
quantitatively. 12

13. 13

14. 14

15. 15

16. 16

17. 17

18. 18

19. CHROMATOGRAPHY
Affinity chromatography
19

20. Biomolecules are purified using purification
techniques that separate according to differences
in specific properties.
INTRODUCTION
• Property Technique*
• Biorecognition Affinity chromatography
(ligand specificity)
• Charge Ion exchange
chromatography
• Size Gel filtration (sometimes
called size exclusion)
• Hydrophobicity Hydrophobic interaction
chromatography
Reversed phase chromatography
20

21. Proteins can be purified from crude sample without the need
for separate clarification, concentration and initial purification
to remove particulate matter.
The adsorbents, used for expanded bed adsorption, capture
the target molecules using the same principles as affinity, ion
exchange or hydrophobic interaction chromatography
21

22. Affinity chromatography separates proteins on the
basis of a reversible interaction between a protein
(and group of proteins) and a specific ligand coupled
to a chromatographic matrix.
Purification that would otherwise be time-consuming,
difficult or even impossible using other techniques
can often be easily achieved with affinity
chromatography.
The technique can be used to separate active
biomolecules from denatured or functionally different
forms, to isolate pure substances present at low
concentration in large volumes of crude sample and
also to remove specific contaminants. 22

23. • Affinity chromatography is a type of liquid
chromatography that makes use of biological
interactions for the separation and specific analysis
of sample components.
• Examples of these interactions include binding of
- an enzyme with an inhibitor
- an antibody with an antigen.
23

24. Principle
• The process itself can be thought of as an
entrapment, with the target molecule becoming
trapped on a solid or stationary phase or
medium.
• The other molecules in the mobile phase will not
become trapped as they do not possess this
property. The stationary phase can then be
removed from the mixture, washed and the
target molecule released from the entrapment in
a process known as dialysis.
24

25. Step’s in Affinity chromatography (purification)
25

26. 26

27. Graphical representation of affinity
chromatography process.
27

28. Common terms in affinity chromatography
Matrix: for ligand attachment. 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 or group of target molecules.
28

29. • Binding: buffer conditions are optimized to ensure that the target
molecules interact effectively with the ligand and are retained by
the affinity medium as all other molecules wash through the
column.
• Elution: buffer conditions are changed to reverse (weaken) the
interaction between the target molecules and the ligand so that
the target molecules can be eluted from the column.
• Wash: buffer conditions that wash unbound substances from the
column without eluting the target molecules or that re-equilibrate
the column back to the starting conditions (in most cases the
binding buffer is used as a wash buffer).
• Ligand coupling: covalent attachment of a ligand to a suitable pre-
activated matrix to create an affinity medium.
• Pre-activated matrices: matrices which have been chemically
modified to facilitate the coupling of specific types of ligand.
29

30. Components in affinity medium
Matrix: for ligand attachment. 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 or group of target molecules.
30

31. 31

32. The matrix ( support material)
The matrix is an inert support to which a ligand
can be directly or indirectly coupled.
It is typically a gel matrix, often of agarose; a linear
sugar molecule derived from algae.
• Traditionally, affinity chromatography support
materials have consisted of porous support
materials such as agarose, poly-methacrylate,
polyacrylamide, cellulose, and silica.
• Other types of support materials are being
developed including nonporous supports,
membranes, flow-through beads (perfusion media),
monolithic supports, and expanded-bed
adsorbents. 32

33. • Nonporous support materials consist of nonporous beads
with diameters of 1- 3 μm.
• These supports allow for fast purifications, but suffer from
low surface areas when compared to traditional porous
supports.
• Membranes used in affinity chromatography also lack
diffusion pores which limits surface area, but like the
nonporous beads allow for fast separations.
• Flow-through beads or perfusion media (originally
developed for ion-exchange chromatography) have both small
and large pores present. The addition of the large flow
through pores allows substances to be directly transported to
the interior of the particle which means only short distances
are required for diffusion.
33

34. • Monolithic supports are based on the same
principle as perfusion media – they contain both
large flow-through pores and small diffusion pores.
• Expanded-bed adsorbents were designed to
prevent column clogging and utilize a reverse in
flow to allow for the expansion of the column bed
which allows for particulates to flow freely through
the column and prevent column fouling.
34

35. Expanded-bed chromatography.
• In this type of chromatography, elution is
performed in a normal
packed-bed, but during the
adsorption-wash step,
the flow is reversed and the
column bed expanded.
• This allows for particulate contaminates to pass
freely through the column and prevent column
clogging.
35

36. The list below highlights many of the properties
required for an efficient and effective
chromatographic matrix.
• Extremely low non-specific adsorption, essential
since the success of affinity chromatography relies
on specific interactions.
• Hydroxyl groups on the sugar residues are easily
derivatized for covalent attachment of a ligand,
providing an ideal platform for the development of
affinity media.
36

37. • An open pore structure ensures high capacity binding
even for large biomolecules, since the interior of the
matrix is available for ligand attachment.
• Good flow properties for rapid separation.
• Stability under a range of experimental conditions
such as high and low pH, detergents and dissociating
agents.
37

38. • Particle size is an additional consideration when
choosing a support material.
• Ideally, small particle sizes are desired to limit mass
transfer effects and limit band broadening.
Unfortunately, as particle size is decreased, backpressures
are increased.
In addition, when using smaller particles, the potential for
the build-up of particulate contaminants and column
fouling is increased.
• For this reason, in preparative applications large
particles (30 –100 μm) are often used.
38

39. • Pore size is another item that must be considered
when using affinity chromatography since,
-- the biomolecules of interest must be able to
not only pass through the column but also be able to
fully interact with the affinity ligand.
The pore diameter should be,
• at least 5 times the diameter of the biomolecule
being purified.
Therefore, a typical protein with a 60 Å
diameter would need a support with at least a 300 Å
39

40. Spacer arm
An important, but often overlooked, aspect in
construction of chromatographic support is that of
the spacer arm.
Spacer arm provides some physical distance b/n the
stationary matrix and the ligand.
Spacer arm are usually long carbon chain with the
general structure,
𝐻2N-(C𝐻2) 𝑛-X, where X = -COOH or -N𝐻2
n = length of carbon chain
between 2 to 12 40

41. 41

42. Spacer arms
• Due to the fact that 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.
42

43. • 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.
43

44. Ligands
44

45. Ligand
• Definition
• noun, plural: ligands
• (1) Any substance (e.g. hormone, drug, functional
group, etc.) that binds specifically and reversibly to
another chemical entity to form a larger complex.
Supplement
• A ligand may function as agonist or antagonist.
• Many ligands are found in biological systems, such
as cofactors and porphyrin in haemoglobin.
Word origin: From Latin ligandus, ‘to bind’.
45

46. Ligands used in affinity
chromatography
• Antibodies.
- polyclonal.
- monoclonal.
• Dye ligands.
• Bio-mimetic dye ligand.
• DNA affinity ligands.
• RNA affinity ligands.
• Peptide affinity ligands.
Etc…
46

47. Antibodies
• Antibodies or immunoglobulins are a type of
glycoprotein produced when a body’s immune
system responds to a foreign agent or antigen.
• Due to the variability of the amino acid sequence in
the antibody binding sites it has been estimated
that antibodies can be produced for millions or
even billions of different foreign agents.
47

48. Typical structure of an antibody.
• The amino acids in the Fc region generally have the
same sequence, whereas the amino acids in the Fab
region have variable amino acid sequences which
allows for the specificity of the binding interaction
against a wide range of antigens.
48

49. Dye-ligand
• Dye ligand chromatography originated in 1968 when
Haeckel et al. were purifying pyruvate kinase using gel
filtration chromatography.
• They found that Blue Dextran (a small dye molecule)
co-eluted with the protein (Haeckel et al, 1968).
• After further investigation, it was determined that
binding between the dye and enzyme caused this
coelution.
• The dye-enzyme binding was later utilized in the
purification of pyruvate kinase using a Blue Dextran
column in 1971.
49

50. Bio-mimetic dye-ligand
• Biomimetic dye-ligand chromatography takes dye-
ligand chromatography one step further and utilizes
modified dyes which mimic the natural receptor of
the target protein.
• In addition to offering better binding affinities,
these modified dyes were initially developed as a
result of the concerns over purity, leakage, and
toxicity of the original commercial dyes.
50

51. • Cibacron Blue 3GA is one of the most common modified
triazine dyes that has been used for protein purification.
• Its structure can be seen in Figure below.
• Chemical structure of blue sepharose dye-ligand
(Cibacron Blue 3GA) commonly used for purification
of albumin as well as enzymes.
51

52. • Covalent attachment of the dye can be achieved
through nucleophilic displacement of the dye’s
chlorine atom by hydroxyl groups on the support’s
surface.
• The main advantages of using dye-ligands and
biomimetic dye-ligands are their low cost and
resistance to chemical and biological degradation.
• The main disadvantage of these synthetic ligands is
that the selection process for a particular
biomolecule is empirical and requires extensive
screening processes during method development.
52

53. DNA affinity ligand
• DNA can also be used as an affinity ligand.
• It can be used to purify DNA-binding proteins, DNA
repair proteins, primases, helicases, polymerases, and
restriction enzymes.
• The scope of biomolecules which can be purified using
DNA is expanded when aptamers are utilized.
• Aptamers are single-stranded oligonucleotides which
have a high affinity for a target molecule.
• SELEX (Systematic Evolution of Ligands by Exponential
Enrichment) allows for the isolation of these
oligonucleotide sequences and allows for a wide range
of potential targets including biomolecules which
typically have no affinity for DNA or RNA. 53

54. Immobilization of affinity ligands
When performing affinity purifications, it is
important to ensure that the affinity ligands are
immobilized so that the binding regions are
exposed and free to interact and bind with the
target molecule(s).
54

55. Activity of the affinity ligand can be affected
by,
• multi-site attachment,
• orientation of the affinity ligand, and
• steric hindrance.
55

56. • When immobilizing antibodies, covalent attachment is
often directed toward the carbohydrate moieties
within the Fc region of the antibody.
• Not only does this limit the number of attachment
sites, but it can also help direct the binding and, thus,
help orientate the antibody so the binding regions
(Fab) are exposed.
• Another way to prevent multi-site attachment is to
- use a support that has a limited number of
reactive sites.
By limiting the reactive sites, the potential for multiple
attachments from a single affinity ligand is greatly
reduced.
56

57. 57

58. Immobilization techniques
• Covalent immobilization.
• Adsorption of ligands.
- non-specific.
- bio-specific.
• Entrapment of ligands.
• Sol-gel entrapment of ligands.
58

59. Covalent immobilization
• 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 onto
support materials.
59

60. Chemical amination of Rhizopus oryzae lipase for multipoint covalent
immobilization on epoxy-functionalized supports
60

61. • Covalent attachment is often directed toward the
carbohydrate moieties within the Fc region of the
antibody leaving behind the binding regions (Fab)
exposed.
• While this may lead to a greater initial cost of
preparation, the stability of these supports
typically is greater and the support does not need
to be periodically regenerated with additional
affinity ligands as is typically the case when using
adsorption techniques.
• As a result, covalent immobilization may be more
economical in the long-term for the immobilization
of costly affinity ligands.
61

62. Adsorption of ligands
• Adsorption of affinity ligands may also be used to
immobilize affinity ligands onto support materials.
• The adsorption can be either,
- nonspecific or
- specific.
• 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. 62

63. 63

64. • Bio specific adsorption 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.
• Both of these immobilization methods allow for
site-specific attachment of the affinity Ligand
which minimizes binding site blockages.
• When bispecific adsorption is used for
immobilization,
the primary ligand (i.e. avidin, streptavidin,
protein A or protein G) must first be immobilized
onto the support material. 64

65. Entrapment of ligands
• Entrapment of affinity ligands was demonstrated
by Jackson et al. when human serum albumin
(HSA) was entrapped using hydrazide-activated
supports and oxidized glycogen.
• This type of immobilization method is generally
less harsh than other immobilization methods and
does not require the use of recombinant proteins.
• In addition, no linkage exists between the affinity
ligand and the support which eliminates the
potential immobilization problems seen before.
65

66. 66

67. Sol-gel entrapment
• Sol-gel entrapment is another method of
encapsulation of affinity ligands.
• The sol-gel entrapment process is as follows:
- First, the sol is formed from a silica precursor
(e.g. alkoxysilane or glycerated silane).
- Once the sol has been formed, the buffered
protein solution is added and the gelation reaction
initiated.
- This is followed by an aging process in which the
sol-gel is dried and further crosslinking of the silica
occurs leaving the protein physically trapped within the
cross-linked silica gel.
67

68. 68

69. Sample preparation and application
• Samples should be clear and free from
particulate matter.
• Simple steps to clarify a sample before
beginning purification will avoid clogging the
column, may reduce the need for stringent
washing procedures and can extend the life of
the chromatographic medium.
69

70. If possible, test the affinity of the ligand: target molecule
interaction.
Too low affinity will result in poor yields since the target
protein may wash through or leak from the column during
sample application.
Too high affinity will result in low yields since the target
molecule may not dissociate from the ligand during elution.
Binding of the target protein may be made more efficient by
adjusting the sample to the composition and pH of the
binding buffer: perform a buffer exchange using a desalting
column or dilute in binding buffer
Sample preparation techniques should ensure that
components known to interfere with binding are removed.
70

71. When working with very weak affinity interactions that are
slow to reach equilibrium, it may be useful to stop the flow
after applying the sample to allow more time for the
interaction to take place before continuing to wash the
column. In some cases, applying the sample in aliquots may
be beneficial.
Do not begin elution of target substances until all unbound
material has been washed through the column by the
binding buffer (determined by UV absorbance at 280 nm).
This will improve the purity of the eluted target substance.
71

72. • The column must be pre-equilibrated in binding
buffer before beginning sample application.
• For interactions with strong affinity between the
ligand and the target molecule that quickly reach
equilibrium, samples can be applied at a high flow
rate.
• However, for interactions with weak affinity and/or
slow equilibrium, a lower flow rate should be used.
• The optimal flow rate to achieve efficient binding
may vary according to the specific interaction and
should be determined when necessary.
72

73. ELUTION
73

74. • Elution methods can be either,
- selective or
- non-selective.
Non-selective gradient elution: either pH or ionic
strength is altered. The pH shift elution using dilute
acetic acid or ammonium hydroxide results from a
change in the state of ionization of groups in the
ligand or the macromolecule that are critical to
ligand-macromolecule binding.
74

75. Specific gradient elution: Buffer containing free molecule of
the ligand that will compete for the binding site of the target
molecules.
75

76. • Selective elution methods are applied in
combination with group-specific ligands whereas
non-selective elution methods are used in
combination with highly specific ligands.
• Forces that maintain the complex include
electrostatic interactions, hydrophobic effects and
hydrogen bonding.
• Agents that weaken these interactions may be
expected to function as efficient eluting agents.
76

77. pH elution
• A change in pH alters the degree of ionization of
charged groups on the ligand and/or the bound
protein.
• This change may affect the binding sites directly,
reducing their affinity, or cause indirect changes in
affinity by alterations in conformation.
• The chemical stability of the matrix, ligand and target
protein determines the limit of pH that may be used.
• If low pH must be used, collect fractions into
neutralization buffer such as 1 M Tris-HCl, pH 9 (60–200
μl per ml eluted fraction) to return the fraction to a
neutral pH.
• The column should also be re-equilibrated to neutral
pH immediately. 77

78. 78

79. 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 linear
gradient or in steps.
• Enzymes usually elute at a concentration of 1 M
NaCl or less.
79

80. 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.
• When working with competitive elution the
concentration of competing compound should be
similar to the concentration of the coupled
ligand.
80

81. 81

82. Reduced polarity of eluent
• Conditions are used to lower the polarity of the
eluent promote elution without inactivating the
eluted substances.
ex.; Dioxane (up to 10%) or ethylene glycol (up to
50%) are typical of this type of eluent.
Chaotropic eluents
• If other elution methods fail, deforming buffers, which
alter the structure of proteins, can be used, e.g.
chaotropic agents such as guanidine hydrochloride or
urea.
• Chaotropes should be avoided whenever possible
since they are likely to denature the eluted protein.82

83. Processing steps in lab scale
83

84. Processing steps in large scale
84

85. Affinity chromatography Applications
85

86. Applications and uses of affinity chromatography
1. Immunoglobulin purification (antibody
immobilization)
- 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-hydroxy
succinimide, N,N’-carbonyl diimidazole, tresyl
chloride, or tosyl chloride.
- Alternatively, free amine groups can react with
aldehyde or free epoxy groups on an activated support.86

87. • Antibodies can also be immobilized by adsorbing
them onto secondary ligands.
• For example,
- if an antibody is reacted with hydrazide biotin,
the hydrazide can react with oxidized carbohydrate
residues on the Fc region of the antibody.
The resultant biotinylated antibody can then be
adsorbed onto an avidin or streptavidin affinity
support.
• This type of biotin immobilization allows for site-
specific immobilization of the antibody
87

88. • Alternatively, antibodies can be directly
adsorbed onto a protein A or protein G support
due to the specific interaction of antibodies with
protein A and G.
• Immobilized antibodies on the protein A or G
support can easily be replaced by using a strong
eluent, regenerating the protein A/G, and re-
applying fresh antibodies.
88

89. 2. Lectin Affinity Chromatography
• This form of chromatography is growing in use.
• Lectins are non-immune system proteins such as
glycoproteins. They originate from animals, plants, and
microorganisms and have an affinity for carbohydrate
residues.
For example, they can separate polysaccharides, glyco-
peptides, and oligosaccharides and cells that contain particular
carbohydrate structures.
• Lectins can be used with an agarose support.
• The purification method can also be combined with other
types of analysis equipment such as high-performance liquid
chromatography (HPLC) and mass spectrometry.
• Also, several lectin columns can be combined for the
purification of glycoproteins and glyco-conjugates in an
approach called serial lectin affinity chromatography.
89

90. 3. 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.
90

91. • In general, the most commonly used tags are
glutathione-S-transferase (GST), histidine fusion
(His or polyHis tag) and protein A fusion tags.
• Other types of fusion tags are also available
including maltose-binding protein, thioredoxin.
91

92. 4. GST tagged purification
• Glutathione S-transferase (GST) is a 26 kDa protein (211
amino acids) located in cytosol or mitochondria and
present both in eukaryotes and prokaryotes (e.g.
Schistosoma japonicum).
• The enzymes have various sources both native and
recombinantly expressed by fusion to the N-terminus
of target proteins.
• GST-fusion proteins can also be produced in Escherichia
coli as recombinant proteins.
Separation and purification of GST-tagged proteins is
possible since the GST tag is capable of binding its
substrate, glutathione (tripeptide, Glu-Cys-Gly). 92

93. • 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)
93

94. GST- tagged protein immobilization. 94

95. 5. 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
95

96. • Histidine tags have strong affinity for metal ions
(e.g. Co2+, Ni2+, Cu2+, and Zn2+).
• One of the first support materials used immobilized
iminodiacetic acid which can bind metal ions and
allow for the coordination complex with the His-
tagged protein.
• One difficulty with iminodiacetic acid supports is
the potential for metal ion leaching leading to a
decreased protein yield.
96

97. • Modern support materials, including nickel-
nitrilotriacetic acid (Ni-NTA) and cobalt-
carboxymethylasparate (Co-CMA), show limited
leaching and, therefore, result in more efficient
protein purifications.
97

98. 6. Frontal Analysis Chromatography
• A mobile phase containing a known
concentration of a target is flushed through an
affinity column with an immobilized binding
agent which becomes saturated.
• Readings during the process will help to create a
curve which will have a mean position that can
contribute to the production of the curve.
• This curve will provide a guide that helps
scientists to derive the kind of interactions that
might be happening with other binding agents.
This can be used to help determine the binding
of drugs. 98

99. 99

100. 7. Affinity purification of albumin and
macroglobulin contamination.
8. Biotin and biotinylated molecules
purification.
9. Protein A, G, and L purification.
10. Reversed phase chromatography.
100

101. Steps of a of reversed phase chromatography separation.
101

102. 102

103. Troubleshooting……
• This section focuses on practical problems that
may occur when running a chromatography
column.
• The diagrams following give an indication of how
a chromatogram may deviate from the ideal
during affinity purification and what measures
can be taken to improve the results.
103

104. 1. Target elutes as a sharp peak.
Satisfactory result
• If it is difficult or impossible to retain biological activity when
achieving this result, either new elution conditions or a new ligand
must be found.
• If using low pH for elution, collect the fractions in neutralization
buffer 104

105. 2. Target is a broad, low peak that
elutes while binding buffer is being
applied
• Find better binding conditions.
105

106. 3. Target elutes in a broad, low peak
Try different elution conditions.
If using competitive elution, increase the concentration of the
competitor in the elution buffer.
• Stop flow intermittently during elution to allow time for the
target molecule to elute and so collect the target protein in pulses
(see second figure on next slide).
106

107. • Note: This result may also be seen if the target
protein has denatured and aggregated on the
column or if there is non-specific binding.
107

108. 4. Some of the target molecule elutes as a
broad, low peak while still under binding
conditions
108

109. • Allow time for the sample to bind and/or apply
sample in aliquots, stopping the flow for a few
minutes between each sample application
• (see second figure on previous slide).
109

110. 110

111. 111

112. 112

113. 113

114. 114