p. 6
Strategy 1. Binding and elution of all sample components
Binding is achieved by ch oosing a start b uffer with a low pH f or HiTrap SP
HP or a high pH for HiTrap Q HP. The ionic stren ght should be kept as l ow as
possible to allow all co mponents to bind t o the ionic exchang e (<5 mS/cm).
This results in a concent ration of the targe t substance and a comp lete
picture of the whole sam ple. The drawback of t his strategy is t hat the
binding capacity of the i on exchanger for th e target substance i s dependent
on the amount of contamina nt in the sample. Stro ngly binding conta minants
can also displace boun d target protein if a lar ge volume of sample is l oaded.
Note: Start condit ions are subject t o the stability of t he sample
components.
Strategy 2. Enrichment of target protein
This is achieved by choo sing a start buf fer with a pH optimi zed to allow
maximal binding of targ et protein, and as high a s possible ionic s trength
to suppress binding of sa mple contaminant s. This strateg y results in a
concentration of the tar get substances.
Strategy 3. Binding of sample contaminants
This is achieved by choo sing a start buf fer with a pH and ioni c strength
that promotes the bind ing of some or all conta minating substa nces but
allows the substance o f interest to pass th rough the column. T he drawback
of this approach is that t he target substan ce is not concentrate d and the
sample volume applie d to the ion exchange r is dependent on th e amount of
contaminants in the sam ple.
Start buff er
The concentration of buf fer required to give e ffective pH cont rol varies
with the buffer sys tem. A list of suitab le buffers and su ggested star ting
concentrations is sho wn in Tables 2 and 3, Figs 2 and 3. I n the majority of
cases a concentration of a t least 10 mM is require d to ensure adequate
buffering capacit y. The ionic stren gth of the buffer s hould be kept low
(< 5 mS/cm) so as not to interfe re with sample bin ding. Salts als o play a
role in stabilizing pr otein structure s in solution and i t is important th e ionic
strength should not b e so low that protein d enaturation or pr ecipitation
occurs.