Use purified and conjugated forms of Thermo Scientific Pierce Recombinant Protein G to probe and detect mouse and human antibodies, especially IgG isotypes, in Western blotting, ELISA, IHC and other immunoassay protocols.
Protein G is a bacterial cell wall protein isolated from group G Streptococci. DNA sequencing of native Protein G identifies two IgG-binding domains and sites for albumin and cell surface binding. The albumin and cell surface binding domains have been eliminated from Recombinant Protein G to reduce nonspecific binding and, therefore, can be used to separate IgG from crude samples. Optimal binding occurs at pH 5, although binding is also effective at pH 7.0 to 7.2.
- Contains two Fc-binding domains per protein
- Better than Protein A for mouse (incl. IgG1), human (incl. IgG3) and rat, goat and cow antibodies
- Poorer than Protein A for guinea pig, pig, dog and cat
- Does not bind human IgM, IgD or IgA
Properties of Recombinant Protein G:
- Source: E. coli
- Molecular Weight: ~21,600 (Apparent MW by SDS-PAGE: 32,000)
- Form: salt-free powder
- A280 of 0.1% solution: 1.0
- Isoelectric point (pI): 4.5
Because Protein G has greater affinity than Protein A for most mammalian IgGs, it may be used for the purification of mammalian IgGs that do not bind well to Protein A. Protein G binds with significantly greater capacity than Protein A to several IgG subclasses such as human IgG3, mouse IgG1 and rat IgG2a. However, Protein G does not bind to human IgM, IgD and IgA. Differences in binding characteristics between Protein A and Protein G may be explained by the differing compositions in the IgG-binding sites of each protein. The tertiary structures of these proteins are very similar although their amino acid compositions are significantly different.
There are inconsistencies in reported binding properties of IgG to Protein G. Variations in isolation and manufacturing methods for Protein G may affect IgG binding, partially because there are differing numbers of IgG-binding sites on various sources of Protein G. Binding studies have been performed using native Protein G and several different recombinant forms. Several assay methods have been used to determine relative affinity, including radiolabeling experiments and ELISA techniques. The differing affinity assays may explain some of the inconsistencies. In addition, there are significant binding differences when different buffers are used. Approximately 44% more IgG from rat serum bound to Protein G when Protein G Binding Buffer was used as compared with 20 mM Tris Buffer, pH 7.5.
- Akerstrom, B. and Bjorck, L. (1986). A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties. J. Biol. Chem. 261(22):10240-7.
- Akerstrom, B., et al. (1985). Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J. Immunol. 135(4):2589 92.
- Bjorck, L. and Kronvall, G. (1984). Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J. Immunol. 133(2):969 74.
- Eliasson, M., et al. (1988). Chimeric IgG-binding receptors engineered from staphylococcal protein A and streptococcal protein G. J. Biol. Chem. 263(9): 4323-7.
- Akerstrom, B., et al. (1987). Definition of IgG- and albumin-binding regions of streptococcal protein G. J. Biol. Chem. 262(28):13388-91.
- Sjobring, U., et al. (1988). Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G. J. Immunol. 140(5):1595-9.
- Fahnestock, S. (1987). Cloned streptococcal protein G genes. Trends in Biochem. Sci. 5:79-83.
- Olsson, A., et al. (1987). Structure and evolution of the repetitive gene encoding streptococcal protein G. Eur. J. Biochem. 168(2):319-24.
- Fahnestock, S.R., et al. (1986). Gene for an immunoglobulin-binding protein from a group G streptococcus. J. Bacteriol. 167(3):870-80.
- Guss, B., et al. (1986). Structure of the IgG-binding regions of streptococcal protein G. EMBO. J. 5(7):1567-75.
- Faulmann, E.L., et al. (1989). Immunological applications of type III Fc binding proteins. Comparison of different sources of protein G. J. Immunol. Methods 123(2):269-81.
Comparison of Protein A, G, A/G and L
Binding characteristics of Protein A, Protein G, Protein A/G and Protein L (Tech Tip #34)
Protein G Agarose Resins, Columns and Kits