Biomedical Engineering Reference
In-Depth Information
µ
µ
l
sample loop. Inject and start the gradient program. The gradi-
ent program may be, for example, 0 - 30 min of 0 - 80% Soln. B in
Soln. A, 5 min 100% Soln. B, 15 min 0% Soln. B (= 100% Soln. A). For
enhanced resolution, steepness of the gradient and concentration
range (%B) may be changed.
Serum albumin will be eluted at the given conditions at about
24 min. To detect very similar conjugation products modify the
gradient: 0 - 10 min 0
Filter the sample using a 0.45-
m syringe filter and fill a 20-
→
30% B, 10 - 20 min 30
→
50% B, 30 -
40 min 50
80% B, 40 - 45 min 100% B, and 45 - 60 min 0% B.
Monitor proteins at 225 nm or at the wavelength of the absorption
maximum of the respective hapten.
Run the respective gradient program from time to time without
protein to check for impurities. Inject about 2 ml 0.1 N NaOH to
remove residual protein and to clean the columns and tubings.
→
3.6 Affinity Chromatography (AC)
Affinity chromatographic purification uses the more or less spe-
cific interactions between macromolecules or between macro-
molecules and low molar mass substances. The binding of a dis-
solved molecule (ligate) to an immobilized ligand should be not too
strong, on one hand, because it has to be eluted without destruction,
and on the other hand, it has to be very selective. Ligand-ligate pairs
with very low dissociation constants K
D
are not useful in affinity
chromatography.
If proteins, e.g., antibodies, are used as immobilized ligands,
medium ligand densities, e.g., 0.5 - 1 mg per milliliter of gel, give
mostly better results than gels with a high amount of immobilized
protein. Reasons for this observation might be steric hindrance
between ligand molecules and/or between neighboring ligands and
ligate as well as unspecific interactions between the ligand and non-
ligate proteins of the sample. Tailing, i.e., the distance b of an elution
peak is significantly larger than distance a (Fig. 3.5), or incomplete
separations indicate such effects.
Since the binding sites of macromolecules are not always located
Immobilization
via spacer
on the surface, interactions between fixed ligands and these sites
may be complicated or inhibited. Chemical modification of the
ligand often overcomes these effects. So the introduction of a spatial
distance between support surface and ligand by an inert spacer
molecule often enables an binding of the ligate. But this spacer
may also introduce hydrophobic or ion exchange effects or form
clusters, which make interactions between ligate and ligand more
difficult.
The first step in affinity chromatography is mostly the prepa-
ration of the affinity matrix by covalent immobilization (coupling)
