Affinity Chromatography - Department of Molecular and Cellular ...
Affinity Chromatography - Department of Molecular and Cellular ...
Affinity Chromatography - Department of Molecular and Cellular ...
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Media characteristics<br />
Lig<strong>and</strong> Composition pH stability* Mean particle<br />
density<br />
size<br />
HiTrap Protein G HP 2 mg/ml Lig<strong>and</strong> coupled to Long term 3–9 34 µm<br />
(MAbTrap Kit) Sepharose HP by Short term 2–9<br />
N-hydroxysuccinimide<br />
activation (gives stable<br />
attachment through<br />
alkylamine <strong>and</strong> ether<br />
links).<br />
Protein G Sepharose 4 2 mg/ml Lig<strong>and</strong> coupled to Long term 3–9 90 µm<br />
Fast Flow Sepharose 4 Fast Flow Short term 2–9<br />
by cyanogen bromide<br />
activation.<br />
*Long term refers to the pH interval over which the medium is stable over a long period <strong>of</strong> time without adverse effects<br />
on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place<br />
<strong>and</strong> sanitization procedures.<br />
Chemical stability<br />
Stable in all common aqueous buffers.<br />
Storage<br />
Wash media <strong>and</strong> columns with 20% ethanol (use approximately 5 column volumes for<br />
packed media) <strong>and</strong> store at +4 to +8 °C.<br />
HiTrap Protein A HP, Protein A Sepharose 4 Fast Flow, HiTrap rProtein A FF,<br />
rProtein A Sepharose 4 Fast Flow<br />
Protein A is derived from a strain <strong>of</strong> Staphylococcus aureus <strong>and</strong> contains five regions that<br />
bind to the Fc region <strong>of</strong> IgG. As an affinity lig<strong>and</strong>, protein A is coupled to Sepharose so<br />
that these regions are free to bind IgG. One molecule <strong>of</strong> protein A can bind at least two<br />
molecules <strong>of</strong> IgG.<br />
Both protein A <strong>and</strong> a recombinant protein A are available from Amersham Pharmacia<br />
Biotech. These molecules share similar specificities for the Fc region <strong>of</strong> IgG, but the<br />
recombinant protein A has been engineered to include a C-terminal cysteine that enables a<br />
single-point coupling to Sepharose. Single point coupling <strong>of</strong>ten results in an enhanced<br />
binding capacity.<br />
The binding strength <strong>of</strong> protein A for IgG depends on the source species <strong>of</strong> the immunoglobulin<br />
as well as the subclass <strong>of</strong> IgG (see Table 2). The dynamic binding capacity depends<br />
on the binding strength <strong>and</strong> also on several other factors, such as flow rate during sample<br />
application.<br />
Although IgG is the major reactive human immunoglobulin, some other types have also<br />
been demonstrated to bind to protein A. Interaction takes place with human colostral<br />
IgA as well as human myeloma IgA 2 but not IgA 1 . Some human monoclonal IgMs <strong>and</strong><br />
some IgMs from normal <strong>and</strong> macroglobulinaemic sera can bind to protein A.<br />
Leakage <strong>of</strong> lig<strong>and</strong>s from an affinity medium is always a possibility, especially if harsh elution<br />
conditions are used. The multi-point attachment <strong>of</strong> protein A to Sepharose results in very<br />
low leakage levels over a wide range <strong>of</strong> elution conditions.<br />
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