This Thermo Scientific NAb Protein A/G Spin Kit is convenient for rapid, small-scale affinity purification of antibodies from a variety of sample types. Included in each kit are sufficient NAb Protein A/G Spin Columns, collection tubes, buffers and a streamlined protocol for purifying at least 2 antibody samples.
Features of Pierce Protein A/G Agarose:
• Protein A/G – immobilized recombinant fusion protein of the antibody-binding domains of Protein A and Protein G enables polyclonal IgG purification from nearly any mammalian species • Agarose resin – support is crosslinked 6% beaded agarose (CL-6B), the most popular resin for protein affinity purification methods • Inert and stable – superior manufacturing method immobilizes Protein A/G by charge-free, leach-resistant covalent bonds, resulting in low nonspecific binding and enabling multiple uses without decline in yield • Standard capacity – Pierce Protein A/G Agarose has a normal load of immobilized Protein A/G, providing a binding capacity greater than 7 mg human IgG/mL resin
Pierce Protein A/G Agarose consists of purified Protein A/G recombinant fusion protein that has been covalently immobilized onto high-quality crosslinked 6% beaded agarose (CL-6B). This particular variety of the resin provides the most versatile combination of chromatographic features for high yield and high purity purification of whole IgG from mammalian serum samples. The agarose beads have physical and chemical properties suitable for many affinity purification systems.
Protein A/G is a genetically-engineered protein that combines the IgG binding domains of both Protein A and Protein G. The fusion protein is expressed in E. coli. Protein A/G contains four Fc binding domains from Protein A and two from Protein G, resulting in a final mass of 50,460 daltons (40 to 45kDa by SDS-PAGE). Protein A/G is not as pH-dependent as Protein A alone, but otherwise has the additive properties of Protein A and G.
Protein A/G binds to all human IgG subclasses, making it the ideal choice for purification of polyclonal or monoclonal IgG antibodies whose subclass identities have not been determined. In addition, it binds to IgA, IgE, IgM and (to a lesser extent) IgD. Protein A/G also binds well to all mouse IgG subclasses but does not bind mouse IgA, IgM or serum albumin. This makes Protein A/G an excellent tool for purification and detection of mouse monoclonal antibodies from IgG subclasses, without interference from IgA, IgM and murine serum albumin. Individual subclasses of mouse monoclonals are likely to have a stronger affinity to the chimeric Protein A/G than to either Protein A or Protein G.
Pierce Protein A/G Agarose is prepared using Thermo Scientific AminoLink Coupling Chemistry, which provides several advantages compared to traditional methods of ligand immobilization. AminoLink Immobilization results in conjugation between sugar monomers of the agarose beads and native lysine residues on the Protein A via simple amide bonds. Unlike typical cyanogen bromide (CNBr) immobilization, the AminoLink Method does not introduce any novel chemical groups that could cause undesired nonspecific binding and produces a stable, essentially irreversible bond. The result is a high-binding-capacity resin that retains functional immobilized Protein A/G through multiple rounds of antibody purification.
Pierce Protein A/G Agarose binds nearly all isotypes and mammalian species of IgG from serum, ascites fluid, cell culture supernantant and other antibody samples, thereby enabling effective antibody purification. Because the immobilized Protein A/G combines the immunoglobulin-binding domains of both Protein A and Protein G, the resin is effective for affinity purification of IgG antibodies from a broad range mammalian host species.
Properties of crosslinked 6% beaded agarose (CL-6B): • Support pH Stability: 2 to 14 (short term); 3 to 13 (long term) • Average Particle Size: 45 to 165 microns • Exclusion Limit: 10,000 to 4,000,000 daltons • Maximum Volumetric Flow Rate: approx. 1 mL/minute (for 1 cm diameter column) • Maximum Linear Velocity: 30 cm per hour • Maximum Pressure: less than 25psi (1.5 bar), defined as the maximum pressure drop across a column that the resin can withstand (Note: The indicated gauge pressure of a liquid chromatography apparatus may be measuring the total system pressure rather than the pressure drop across the column.)