Affinity

Chromatography
PLUS VIKA SEPTIDEANI
M. ADAM FIRLIANSYAH
 The goal of affinity chromatography is to
separate all the molecules of a particular
specificity from the whole gamut of
molecules in a mixture such as a blood
serum. For example, the antibodies in a
serum sample specific for a particular
antigenic determinant can be isolated by
the use of affinity chromatography.
Step 1.
An immunoadsorbent
is prepared. This
consists of a solid matrix
to which the antigen
(shown in blue) has
been coupled (usually
covalently).
Agarose, sephadex,
derivatives of cellulose,
or other polymers can be
used as the matrix.
Step 2.

The serum is passed over the

immunoadsorbent. As long as the
capacity of the column is not exceeded,
those antibodies in the mixture specific
for the antigen (shown in red) will bind
(noncovalently) and be retained.
Antibodies of other specificities (green)
and other serum proteins (yellow) will
pass through unimpeded.
Step 3.
Elution. A reagent is passed into the column to release
the antibodies from the immunoadsorbent. Buffers
containing a high concentration of salts and/or low pH
are often used to disrupt the noncovalent 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, gentler, approach is to elute with a soluble
form of the antigen. These compete with the
immunoadsorbent for the antigen-binding sites of the
antibodies and release the antibodies to the fluid
phase.
 Step 4.
Dialysis. The eluate
is then dialyzed
against, for example,
buffered saline in
order to remove the
reagent used for
elution.
Isolating
Transcription
Factors
 Transcription factors are extraordinarily diverse, but
any one factor represents only a tiny fraction of the
protein molecules present in the cell. This page
describes how one can isolate and purify such rare
molecules.
Example: isolating the lac repressor
An E. coli cell contains only 10-20 copies of the lac
repressor. This represents a ratio of only 1 molecule in
50,000 protein molecules in the cell.
However, the specificity of the lac repressor for the
DNA sequence of the operator provides a mechanism
for fishing it out of the mixture.
 The procedure:
Learn the sequence to which the repressor
binds (by footprinting).
Synthesize a segment of DNA containing the
sequence.
Attach this artificial molecule to beads of an
inert, solid medium (the matrix).
Pour an extract of E. coli cells over the beads.
Only molecules specific for the DNA sequence
in this case, molecules of the lac repressor
will bind to the beads.
After irrelevant protein molecules have passed
through the column,
wash the beads with a buffer that will release
the lac repressor molecules so they can be
studied
This technique is called affinity chromatography.
Many eukaryotic transcription factors have
also been isolated and studied by this method.