Biomedical Engineering Reference
In-Depth Information
and wash until the conductance of the eluate has the value of the
starting buffer (eluent).
3.4.2 Capacity Test
The capacity indicated in Table 3.2 is for orientation only and differs
from protein to protein; therefore, check the capacity in an assay
with the same conditions as for separation and monitoring.
The decision on use of an anion or cation exchange medium
depends on binding of the protein of interest or binding of accom-
panying substances to the matrix. If the protein, which should be
absorbed, has an isoelectric point (pI) below pH 7 (acidic protein),
select an anion exchanger. If the absorbed molecule carries only
few charged groups on its surface, a strong exchange medium is
recommended (SP and QA). If the macromolecule carries a high
charge density or if desorption (elution) with smooth conditions
is favored, select a weak ion exchange medium (CM and DEAE).
For binding choose a buffer with low ionic strength and pH at
least one unit below pI for cation exchange and one pH unit above
pI for anion exchange, respectively.
If the pH dependency of biological stability and/or activity is
known, a cation exchanger is recommended if this stability is below
the pI.
Check the conditions for binding (adsorption) to the IEC gel as
follows:
Equilibrate a series of 0.1 - 0.5 ml of ion exchange medium in
test tubes with 50 mM buffer without any additional neutral salt.
The buffers should differ in pH for 0.5 units each.
Add 0.5 - 1 ml of protein sample, dialyzed against the respective
buffer, to the wet media. Incubate for 15 - 30 min at that temperature
which should be used for separation. Separate by centrifugation
or filtration after incubation and determine the amount of target
protein in the liquid. For binding buffer select that buffer at which
the target protein disappears.
The capacity of the exchange medium is determined with a se-
ries of protein dilution in binding buffer and a constant amount of
IEC gel.
For preparative separations, take a gel volume such that its
capacity is required for only two-thirds of maximal binding.
3.4.3 Sample Application
The binding of proteins to IEC media follows the law of mass action;
therefore, complete binding is not possible; bound and free proteins
are in equilibrium.
Loading of supports can be done in two ways: batch loading
and column loading.
