Performing a Purification of IgG Antibodies with MabTrap® Kit

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

MAbTrap® Kit contains one HiTrap® Protein G HP 1 ml column, binding, elution, and neutralization buffers, a syringe with fittings, and an optimized purification protocol (Figure 3.12). The kit contains sufficient material for up to 20 purifications of monoclonal or polyclonal IgG from serum, cell culture supernatant, or ascites, when using a syringe. The column can also be connected to a peristaltic pump if preferred.

MabTrap Kit

Figure 3.12. MabTrap® Kit contains both a 1 ml HiTrap® Protein G HP column and premade buffers for antibody purification.

 

Figure 3.13 shows the purification of mouse monoclonal IgG1 from cell culture supernatant using a syringe operation and a similar purification with pump operation. Operating HiTrap® Protein G HP 1 ml with a syringe resulted in an IgG1 pool of 3 ml with an absorbance of 0.44 (A280) and a corresponding yield of 0.9 mg pure mouse monoclonal IgG1. A similar experiment in which the column was operated with a P-1 pump resulted in an IgG1 pool of 2 ml with an absorbance of 0.60 (A280), corresponding also to a total yield of 0.9 mg pure mouse monoclonal IgG1.

Purification of mouse monoclonal IgG1 from cell culture supernatant

Figure 3.13. Purification of mouse monoclonal IgG1 from cell culture supernatant; (A) using a syringe and (B) a peristaltic pump. (C) Analysis of eluted fractions by nonreducing SDS-PAGE on PhastSystem using PhastGel 10–15, silver stained.

 

Sample preparation, purification, and storage

Refer to HiTrap® Protein G HP columns regarding sample preparation, purification protocol, and storage recommendations.

 

Buffer preparation

Binding buffer: Dilute binding buffer concentrate 10-fold

Elution buffer: Dilute elution buffer concentrate 10-fold

Neutralizing buffer: 1 M Tris-HCl, pH 9.0

Dilute the 10× buffer concentrates with high quality water as follows:

  1. 2.5 ml binding buffer concentrate + 22.5 ml high quality water to a total volume of 25 ml.
  2. 0.5 ml elution buffer concentrate + 4.5 ml high quality water to a total volume of 5 ml.

 

Materials

     
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