Protein A Chromatography
Protein A affinity chromatography is a rapid one step purification, which removes most non IgG contaminants and can achieve purities close to homogeneity. It is particularly useful for purifications of tissue culture supernatant, where 10–00 fold concentrations can be achieved.
Protein A can withstand more harsh conditions which can be beneficial for deep cleaning and regeneration. Different mouse IgG subclasses will exhibit varying strength of association to Protein A. Customization of the purification strategy may be required for the affinity separation, e.g. mouse IgG1, the most common subclass used, does not bind well to Protein A at low ionic strength. However, the use of high salt concentrations (2–3M NaCl) and high pH (pH 8–9), these antibodies will bind to Protein A and provide good separation.
The needs of the researcher dictate that the speed of sample processing, the cost and the reproducibility are key criteria for selecting purification tools. Amintra® purification resins have been designed to offer the optimal solution to each criterion. In the vast majority of cases, simply selecting the correct resin and performing a considered purification strategy will provide the best possible separation of your target proteins.
Protein A is a cell wall protein from Staphylococcus aureus with a molecular weight between 35–50 kDa. The quality of the Protein A agarose (or equivalent) is important to avoid leakage of Protein A during the elution procedure. Immobilized Protein A resins linked via an amide bond between the amino groups of Protein A and either oxirane or N-hydroxysuccinimide ester groups form the most stable crosslinks. Immobilized Protein A binds specifically to the Fc region of immunoglobulin molecules of many mammalian species.
Supporting matrix: Highly cross-linked 4% agarose supplied as 50% slurry
Ligand: Recombinant Protein A
Bead size range: 45-165 µm
Recommended working pH: 2.5-9.0
Typical binding capacity: >40 mg Human IgG /ml resin
Maximum Flow rate: Up To 300 cm/h
Maximum pressure: 0.3MPa (3 bar)
Chemical stability: High – Stable in all aqueous buffers commonly used in Protein A chromatography:
- 10 mM HCl (pH 2)
- 1 mM NaOH (pH 11)
- 0.1 M sodium citrate/HCl (pH 3)
- 6 M guanidine-HCl
- 20% ethanol
Storage buffer: 1x PBS containing 20% ethanol
Protein A and Protein G Antibody binding affinity: