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Affinity Chromatography
Principle -Affinity chromatography is a highly selective method used for the purification of
specific molecules from complex mixtures based on their binding affinity to a specific ligand
that is immobilized on the stationary phase
...

The principle relies on the specific interaction between a target
molecule (e
...
, a protein) and a ligand that is designed to specifically bind to it
...
The affinity of the interaction is high, so the target molecule will preferentially
bind to the ligand, while other non-target molecules flow through the column
...
The sample is loaded onto a column containing the immobilized ligand
...
The target molecule binds to the ligand due to their high affinity
...
Non-binding molecules are washed away, and the bound target is eluted by changing
the conditions (e
...
, altering pH, salt concentration, or using a competing ligand)
...
Stationary Phase (Column Matrix):
o The stationary phase in affinity chromatography typically consists of a solid
matrix such as agarose, silica, or Sepharose
...

o The stationary phase is functionalized with a specific ligand (e
...
, an antibody,
enzyme substrate, or metal ion) that will selectively bind to the target
molecule
...

2
...
It is immobilized
on the stationary phase, and its specific binding to the target molecule allows
for selective capture
...

3
...

o The elution buffer may contain salt, pH modifiers, or competing ligands that
disrupt the interaction between the ligand and the target molecule, allowing
the target to be released and collected
...
Sample:
o The sample is the mixture that contains the target molecule to be purified
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o

It is typically loaded onto the column after any preliminary steps, such as
filtration, to remove large debris
...
Collector (Fraction Collector):
o The collector is responsible for collecting the fractions that elute from the
column
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Procedureo
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The stationary phase (solid matrix) is functionalized with the chosen ligand
and packed into a chromatography column
...

The sample, containing the mixture of molecules, is applied to the top of the
column
...

After the sample is loaded, the column is washed with an equilibration buffer
to remove non-specifically bound substances
...

To elute the bound target molecule, the conditions of the column are changed
...

The target molecule is then released from the stationary phase and collected in
fractions
...
This involves
removing any remaining bound molecules and resetting the column conditions
to the initial state
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Applications
1
...

o Antibody-based affinity chromatography is used for purifying antigens (e
...
,
to isolate a specific protein or pathogen) or immunoglobulins (antibodies)
...

o Affinity chromatography can also be used to purify enzymes that specifically
bind to their natural substrate
...

o DNA and RNA can be purified using affinity chromatography by designing
specific ligands such as oligonucleotides that bind to complementary

sequences of nucleic acids
...

o Affinity chromatography can be applied to the purification of small molecules
or metabolites that have specific affinity for certain ligands, such as drugs, cofactors, or other bioactive compounds
...
Clinical Diagnostics: In clinical settings, affinity chromatography is used for isolating
biomarker molecules or purifying diagnostic proteins
...

3
...
It is also used in the screening of potential drug candidates that
bind to specific biological targets, such as enzymes or receptors
...
Purification of Growth Factors: Growth factors or cytokines can be purified from
culture media by using affinity chromatography with ligands that selectively bind
these molecules
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