Recombinant tagged proteins containing a protein A tag can be puriﬁed on IgG Sepharose® 6 Fast Flow. The AC medium is based on the Sepharose® 6 Fast Flow matrix, with human IgG covalently coupled to it. The mechanical characteristics of this Fast Flow medium allows high ﬂow velocities (up to 400 cm/h) to be used for rapid and convenient single-step puriﬁcation of protein A-tagged protein conjugates produced in prokaryotic expression systems. Up to 2 mg/ml protein A can be bound to the ligand at pH 7.5.
IgG Sepharose® 6 Fast Flow is also suitable for tandem afﬁnity puriﬁcation (TAP) of protein complexes.
An alternative eluent is 0.1 M glycine-HCl, pH 3.0. Chaotropic agents may also be used for elution.
Characteristics of IgG Sepharose® 4 Fast Flow are shown in Table 1.
* Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance.
* Since elution conditions are quite harsh, it is recommended to collect fractions into neutralization buffer (60 µL – 200 µL 1 M Tris-HCl, pH 9.0 per ml fraction), so that the ﬁnal pH of the fractions will be approximately neutral.
This method, while giving a concentrated eluate, can only be used if the fusion product is stable under the acid conditions.
Figure 1.Chromatogram of a sample taken at one time point during fermentation. (B) Results from automatic monitoring of the product concentration during fermentation. Concentration of ZZ-IGF-1 is determined by integration of the ZZ-IGF-1 peak obtained during each chromatographic analysis. Bacterial density is measured manually at A600 nm.
Avoid reducing agents such as 2-mercaptoethanol or DTT since they may disrupt disulﬁde bonds within the IgG ligand.
Wash with 5 column volumes of 20% ethanol at neutral pH and store at +4 °C to +8 °C.