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Strategy 1. Binding and elution of all sample components
Binding is achieved by choosing a start buffer with a low pH for HiTrap SP
HP or a high pH for HiTrap Q HP. The ionic strenght should be kept as low as
possible to allow all components to bind to the ionic exchange (<5 mS/cm).
This results in a concentration of the target substance and a complete
picture of the whole sample. The drawback of this strategy is that the
binding capacity of the ion exchanger for the target substance is dependent
on the amount of contaminant in the sample. Strongly binding contaminants
can also displace bound target protein if a large volume of sample is loaded.
Note: Start conditions are subject to the stability of the sample
components.
Strategy 2. Enrichment of target protein
This is achieved by choosing a start buffer with a pH optimized to allow
maximal binding of target protein, and as high as possible ionic strength
to suppress binding of sample contaminants. This strategy results in a
concentration of the target substances.
Strategy 3. Binding of sample contaminants
This is achieved by choosing a start buffer with a pH and ionic strength
that promotes the binding of some or all contaminating substances but
allows the substance of interest to pass through the column. The drawback
of this approach is that the target substance is not concentrated and the
sample volume applied to the ion exchanger is dependent on the amount of
contaminants in the sample.
Start buff er
The concentration of buffer required to give effective pH control varies
with the buffer system. A list of suitable buffers and suggested starting
concentrations is shown in Tables 2 and 3, Figs 2 and 3. In the majority of
cases a concentration of at least 10 mM is required to ensure adequate
buffering capacity. The ionic strength of the buffer should be kept low
(< 5 mS/cm) so as not to interfere with sample binding. Salts also play a
role in stabilizing protein structures in solution and it is important the ionic
strength should not be so low that protein denaturation or precipitation
occurs.
