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Affinity Chromatography

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79 views

Affinity Chromatography

Uploaded by

kavya aras
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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AFFINITY CHROMATOGRAPHY

Introduction
•Affinity chromatography was invented in 1968 by Pedro
Cuatrecasas and Meir Wilchek.
•Affinity chromatography is a method of separating biochemical
mixtures, based on a highly specific biological interaction such as
that between antigen and antibody, enzyme and substrate, or
receptor and ligand.
Introduction
 A ligand which exhibits a specific affinity for particular
compound gets covalently bonded to gel matrix and material is
filled into the column.

 Choice of gel depends on type of group present in ligand


molecule and nature of binding reaction with the substance to be
purified.

 Samples are applied under favourable conditions for their


specific binding to the ligand.
Common terms used in AC
Matrix: It is used for ligand attachment. Matrix should be
chemically and physically inert.
Spacer arm: It is 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.
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.
Common terms used in AC
Principle of AC
Affinity Chromatography

• The antibodies in a serum sample specific


for a particular antigenic determinant can be
isolated by the use of affinity
chromatography.
Step-1:
 An immuno adsorbent is prepared.

 This consists of a solid matrix to which the


antigen (shown in blue) has been coupled
(usually covalently).

 Agarose, derivatives of cellulose, or other


polymers can be used as the matrix.
Affinity Chromatography

Step-2:
The serum is passed over the immuno
adsorbent.

The antibodies in the mixture specific for


the antigen (shown in red) will bind and be
retained.

Antibodies of other specificities (green)


and other serum proteins (yellow) will pass
through unimpeded.
Affinity Chromatography
Step-3:
 Elution: A reagent is passed into the column to release the
antibodies from the immuno adsorbent.

 Buffers containing a high concentration of salts and/or low pH are


often used to disrupt the interactions between antibodies and antigen.
A denaturing agent, such as 8 M urea, will also break the
interaction by altering the configuration of the antigen-binding site of
the antibody molecule.

 Another, approach is to elute with a soluble form of the antigen.


These compete with the immuno adsorbent for the antigen-binding
sites of the antibodies and release the antibodies to the fluid phase.
Affinity Chromatography
Step-4:

 Dialysis: The elute is then dialyzed against, for example, buffered


saline in order to remove the reagent used for elution.
12
Affinity Chromatography

Uses:
 Purify and concentrate a substance from a mixture into a buffering
solution.

 Reduce the amount of a substance in a mixture.

 Discern what biological compounds bind to a particular substance.

 Purify and concentrate an enzyme solution.

 Purify certain proteins from a mixture.


Affinity Chromatography
Advantages:

 High degrees of purity can be obtained.


 The process is very reproducible.
 The binding sites of biological molecules can be simply
investigated.

Disadvantages:

 Cost.
 The difficulties associated with scale-up.
 The high labour intensity.
Applications of AC

Affinity chromatography can be used in a number of applications,


including nucleic acid purification, protein purification from cell
free extracts and antibody purification from blood serum.
 The most common use of affinity chromatography is for the
purification of recombinant proteins. It is also known as
recombinant proteins-affinity chromatography.
 This technique is also used for the purification of antigen from
blood serum. It is also known as antigen-affinity chromatography.
Applications of AC

 Purification of proteins

 Tagged protein purification.

 Antibody purification.
 Separation of protein by Lectin affinity chromatography
 It is a form of affinity chromatography where lectins are used
to separate components within the sample.
 Lectins, such as concanavalin A, are proteins which can bind
specific carbohydrate (sugar) molecules.
 The most common application is to separate proteins based on
their glycan groups.
Applications of AC

 Study of drugs

 Melanin binding Interactions.

 Quantitative determination

 Metallothionein (MT) in physiological fluid.


 Affinity chromatography gel is used as a solid phase extraction (SPE)
support for pre concentration of MT Proteins and metals bound to MT
from human urine samples.

 The present method can be used in toxicological analysis for


determination of MT concentration in physiological fluids in subjects
exposed to heavy metals.
Which one among the following is not the uses of
affinity chromatography
a) Purify certain proteins from a mixture
b) Purify and concentrate an enzyme solution
c) Reduce the amount of a substance in a mixture
d) Inorganic compound
d) Inorganic compound
Affinity chromatography was invented in ____ by
Pedro Cuatrecasas and Meir Wilchek
a) 1968
b) 1978
c) 1958
d) 1948
a) 1968
______ is used to improve binding between ligand and
target molecule.
a) Ligand
b) Spacer arm
c) Matrix
d) Ligand coupling
b) Spacer arm

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