View - ResearchGate

Affinity Capillary Electrophoresis 305
and screening for ligand-binding sites, optimization of CE resolution, conformation
structure-function studies, detection of functional heterogeneity and
estimation of quantitative binding parameters such as binding (stability) and
rate constants. The reasons for using CE for such studies will typically be the
scarcity of biological material, the unique ability of CE to be applied to unlabelled
interacting compounds of similar size and the convenient and fast analyses.
An attractive feature of using CE for affinity studies is the versatility, i.e., the
variety of approaches available making it possible to accommodate CE to a wide
range of interactions. The ACE methods may be divided into two major groups:
(1) the mobility shift and (2) the pre-equilibrium (pre-eq) assays. Changes in
analyte mobility as a function of ligand concentration are used for determination
of binding constants in the mobility shift assays. The pre-eq assays are
characterized by introduction of a pre-incubated sample containing both of the
interacting species into a capillary containing only neat electrophoresis buffer.
Upon separation, peak heights or areas are used in the subsequent data analysis.
The main features of the various ACE methods are summarized in Table 1.
Unfortunately, a wealth of different names, acronyms and abbreviations have
been assigned to ACE methods by different groups (see Note 1).
3. Experimental Variables
In any CE experiment, the most important decisions deal with the choice
of capillary column, the washing solutions, the electrophoresis and sample
buffers and the choice of running conditions. In affinity electrophoresis, these
choices are all very dependent on the type of analyte and ligand. For a thorough
evaluation of the relative importance of the parameters affecting the separation
performance of CE experiments, it is worthwhile to consult (21). Factors and
practical considerations that affect molecular interactions, recovery and reproducibility
of peak shape, area and appearance time in the CE analyses of
proteins will be focused on here.
3.1. Capillaries
The standard approach is to use columns of uncoated fused synthetic silica
of 50 m in internal diameter (i.d.) and of 40–70 cm in length. This i.d. in
most instruments gives an appropriate detection path length at the same time
as the induced Joule heating is efficiently removed. In this regard, instruments
equipped with liquid cooling may be advantageous over forced air-cooled
instruments and definitely over instruments with no active cooling (22). Even
though different approaches exist to estimate the distribution of temperature
inside a capillary buffer during a run (21–23), the cooling may not so much
be used to secure a given temperature during an analysis but more to make