Biomedical Engineering Reference
In-Depth Information
Batch loading
Mix the protein dissolved in binding buffer with equilibrated IEC
gel. Agitate for several minutes and separate gel and solution either
by centrifugation or filtration. Wash and elute the gel on a funnel or
in a column. Since the equilibrium is established only once, a nearly
complete absorption occurs if the gel is in a large excess. The
advantage of batch loading is the easy separation of gel and liquid.
Column loading
Prepare a column with the equilibrated gel. In the opposite to GPC
the ratio of diameter to bed height may be 1:1 and the sample
volume may be much larger than the bed volume. A low bed height
has the advantage of little change of height during elution when
buffers with altered ionic strength or pH are used. Furthermore,
the binding equilibrium establishes very often during movement
of the sample through the column.
3.4.4 Elution
Elution of bound material is achieved by changing the buffer com-
Gradient elution
position, i.e., by increasing the ionic strength (addition of neutral
salts as sodium or potassium chloride) or by changing the pH of
the elution buffer. This change may be done by sequential addition
of different elution buffers (stepwise gradient) or by application of
a continous change (continous gradient) produced using a gradient
mixer (cf. Fig. 2.2). In IEC mostly the basic buffer composition is
retained and the ionic strength is increased by increasing the con-
centration of salt. The elution is monitored by UV measurement,
and the development of the gradient is checked by measuring the
conductivity and/or the pH.
It is possible to combine stepwise gradient and continuous gra-
dient, i.e., the starting elution buffer has a higher ionic strength
than the binding buffer, and after a short time the elution of the
protein(s) of interest starts by application of a flat continuous gra-
dient.
When soft IEC media are used, adaptor columns are not suitable
because the volume of the gel alters (mostly shrinks) when salt
concentration increases; hence, the flow direction is from top to
bottom.
Flow rate of the elution buffer as well as change of buffer compo-
sition should not be too fast because the buffer components need
some time to diffuse into the gel and proteins bound within the
pores of the gel have to move outside.
3.4.5 Cleaning and Regeneration
After separation, wash the gel with two bed volumes of 2 M KCl or
6 M guanidinium chloride and re-equilibrate it. If this purification
is not sufficient, cycle the gel as described in Sect. 3.4.1.
