Antibody Selection Guide

Antibody Selection Guide

The principle of antibody affinity chromatography is that Protein A and Protein G have high affinity and specificity for the Fc-region of IgG from different sources. These proteins are immobilized on many of our fillers, creating an excellent method for isolating IgG and IgG subclasses from ascites, cell culture supernatants and serum. For a complete range of antibody affinity purification, see the Antibody Affinity Fillers and Column Selection Guide on the next page.

Protein A (Protein A) is a strain derived from Staphylococcus aureus that contains five domains that specifically bind to the Fc segment of an antibody IgG molecule and specifically binds to antibody molecules in the sample. Other miscellaneous proteins flow through and have extremely high selectivity, usually achieving a purity of more than 95% by one-step affinity chromatography. ProteinA sepharoseCL-4B is a medium obtained by purifying protein A from S. aureus by a method of CNBr coupled to sepharose CL-4B to purify immunoglobulin in body fluid or cell culture medium. nproteinA sepharose4FF, nprteinA is native ProteinA, indicating that it does not introduce any animal-derived components during the production process. rproteinA sepharose4FF, recombinant protein A (rProtein A), genetically engineered to contain a C-terminal cysteine, can be conjugated to the agarose backbone at a single site, effectively reducing steric hindrance and increasing IgG Binding ability. At the same time, the ligand does not cite human IgG during fermentation and purification, avoiding the risk of contamination of human IgG. rmpProteinA, a multi-site attachment technology that ensures lower ligand shedding. Monoclonal and polyclonal antibodies are highly purified in one step.

In 2005, GE introduced a new generation of MabSelect, the first novel protein A chromatography media using high flow Agarose as a backbone, designed for large-scale antibody purification. Compared with traditional Fast Flow packing, high flow rate agarose has high flow rate and high load, lower ligand shedding, easy cleaning and sterilization, longer life and more economical, suitable for rapid and efficient antibody production and amplification. It is the standard medium for purification and amplification of therapeutic monoclonal antibodies. In 2011, the new product MabSelect LX was introduced, with a higher dynamic load of 60 mg Mab/ml, which further reduced the cost.

Protein G is a cell surface protein of Group G Streptococcus, a type III Fc-receptor that binds to IgG similar to Protein A and binds with a non-immune mechanism. We provide recombinant protein forms (HiTrap Protein G HP, Ab SpinTrap, ProteinG Sepharose 4 Fast Flow, GammaBind G Sepharose, and GammaBindPlus Sepharose) that are expressed in E. coli after gene knockout of their native proteins. Protein G contains two Fc binding regions. Ab SpinTrap columns pre-loaded with Protein G SepharoseHigh Performance are also available, which facilitate the capture and purification of monoclonal and polyclonal antibodies by centrifugation.

Although protein A and protein G affinity fillers are similar in many respects, the specificity for IgG is different. A general antibody capture protein G affinity filler is a good choice because it binds to a wide range of eukaryotic organism-expressed IgG and can bind to more IgG subtypes.

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