Performing a Purification of IgG Antibodies with HiTrap® rProtein A FF columns

Extracted from Affinity Chromatography Vol. 1: Antibodies, GE Healthcare, 2016
 

HiTrap rProtein A FF is a ready-to-use column prepacked with rProtein A Sepharose Fast Flow (Figure 3.20) for the convenient purification of monoclonal antibodies from cell culture supernatants, serum, and ascites. The column is suitable for small-scale purification of monoclonal antibodies from multiple species, screening, and process development. The columns are available in 1 ml and 5 ml sizes. In common with all HiTrap columns, HiTrap rProtein A FF can be used for rapid purification using a syringe, pump, or chromatography system. Furthermore, purification capacity can be greatly increased by connecting columns in series.

HiTrap rProtein A FF columns Purification of mouse IgG2b from ascites on a 1 ml HiTrap rProtein A FF column

Figure 3.21. (A) Purification of mouse IgG2b from ascites on a 1 ml HiTrap rProtein A FF column using a syringe. (B) Analysis of eluted IgG2b by SDS-PAGE on PhastSystem and a PhastGel Gradient 10-15 precast gel revealed a purity of 95% (silver staining).

 

Sample preparation, buffer preparation, and purification

HiTrap Protein A columns use the same protocol as HiTrap Protein G HP.

Refer to Table 3.5 regarding binding and elution conditions commonly used with Protein A Sepharose chromatography media for purification of IgG from different species.

Materials

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