Protein G Chromatography
Protein G binds to all major Ig classes except IgM and therefore has a wider reactivity profile than Protein A. However, the binding of Igs to Protein G is often stronger, requiring more stringent elution conditions for complete recovery of the immunoglobulin compared to Protein A.
Amintra® Protein G Magnetic Resin is a fast flow sepharose matrix covalently modified with recombinant Protein G expressed in E. coli. The albumin domain naturally present in these proteins has been deleted to create a resin with a high capacity and affinity for antibodies. Typically binding capacity of 25 mg IgG / ml resin.
Protein A or Protein G Chromatography
Immunoglobulin G from most species consists of several subclasses with different biological properties. Four subclasses of IgG have been identified in human (IgG1, IgG2, IgG3, and IgG4) and in mouse (IgG1, IgG2a, IgG2b and IgG3). For immunological studies, it is often necessary to isolate one particular subclass of IgG from the other subclasses.
Sandwich ELISA assay can also be used to accurately measure antibody concentrations within a range of 1 mg/ml to 20 mg/ml sample. The antibodies can also be monitored for purity by SDS-PAGE under reducing or non-reducing conditions. Note that IgG appears in a reducing SDS-PAGE as 25 kDa and 50-55 kDa bands and IgM appears as 25 kDa and 70-80 kDa bands. Recovery of immunoglobulins can be quantified by Bradford assay, scanning densitometry of reducing or non-reducing SDS-polyacrylamide gels or ELISA.
- Supporting matrix: Highly crosslinked 4% agarose supplied as a 50% slurry
- Ligand: Recombinant Protein G
- Bead size range: 45-165 µm
- pH stability: long term: pH 3 – 9 –short term: pH 2 – 10
- Typical binding capacity: >30 mg Human IgG /ml resin
- Maximum pressure: 0.3MPa (3 bar)
- Chemical stability: High. The IgG binding capacity and recovery was maintained after storage for:
- 7 days at 37° in:
- 1 M acetic acid pH 2.0, 20 mM sodium phosphate, 1% SDS, pH 7.0
- 6 M Guandine-HCl, pH 7.0
- 70% ethanol
- 2 hours at room temperature in:
- 0.1 M HCl, pH 1.0
- 8 M urea, pH 10.5
- 0.1 M Glycine-NaOH, pH 11.0
- 7 days at 37° in:
- Solubility in water: Insoluble
Protein A and Protein G Antibody binding affinity: