Protein G affinity chromatography

L.J. Janis and F.E. Regnier, Dual-column immunoassays using protein G affinity chromatography, Anal. Chem., 61 (1989) 1901-1906. 

Protein A or Protein G affinity chromatography Immunoglobulins show specific affinity for these proteins which can be obtained complexed to Sepharose (Pharmacia). It is a simple matter so apply antiserum diluted in 20 mM phosphate buffer to a small column and subsequently elute the pure IgG using a glycine buffer pH 2.7. 

Bond, A., Jones, M. G., and Hay, F. C. (1993). Human IgG preparations isolated by ion-exchange or protein G affinity chromatography differ in their glycosylation profiles.. Immunol. Methods 166, 27-33. 

H Zou, Y Zhang, P Lu, IS Krull. Characterization of immunochemical reaction for human growth hormone with its monoclonal antibody by perfusion protein G affinity chromatography and capillary zone electrophoresis. Biomed Chro-matogr 10 78-82, 1996. 

Antibody production from 8C2 cells growing within the hollow fiber bioreactor reached 240mg/wk by the third week. ATI was purified from the media through protein G affinity chromatography (Figure 6.5-5). Final ATI concentrations in the... 

Affinity chromatography and related techniques (e.g., thiol chromatography and IMAC) are widely used for preparative isolation because they enable a single protein or class of proteins to be selectively purified from very complex mixtures. They may be occasionally used as analytical tools. For example, protein A affinity chromatography has been used for quantitative analysis of immunoglobulins in ascites fluid.45 Information about surface-accessible histidine and phosphate groups may be obtained using IMAC. 

Q Wang, G Luon, J Ou, WSB Yeung. Noncompetitive immunoassays using protein G affinity capillary chromatography and capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr A 848 139-148, 1999. 

Reactors of this type have been used to produce and harvest the heavy chain of a murine monoclonal antibody using protein G affinity and histidine-tagged human granulocyte-macrophage colony-stimulating factor (GM-CSF) using metal affinity chromatography. Implementation of this scheme on a small scale was difficult, but these studies show that the amount of recoverable protein can be increased [62]. As shown in Fig. 4, recoveries of human GM-CSF and a mouse monoclonal antibody heavy chain were increased by three- and seven-fold, re-... 

An example of virus clearance factors in chromatographic processes frequently used for purification of antibodies is given in Table 17. Lower clearance factors for protein A affinity chromatography have been found by Mariani and Tarditi128 when compared to results found with protein G by Walter and Allgaier.237 An explanation of this fact can be found in the fact that protein G requires harsher elution conditions than protein A. 

Zapata, G. (1999). Recovery of recombinant antibodies from unclarified Chinese hamster ovary cell culture fluid by expanded bed Protein A affinity chromatography. Proc. Waterside Monoclonal Conf. Norfolk, VA. 

Gould, B. J., Hall, P. M., and Cook, J. G. (1984). A sensitive method for the measurement of glycosylated plasma proteins using affinity chromatography. Ann. Clin. Biochem. 21, 16-21. 

Yatscoff RW, Tevaarwerk G), MacDonald JC. Quantification of nonenzymically glycated albumin and total serum protein by affinity chromatography. Clin Chem 1984 30 446-9. 

Fahrner, R., Whitney, D., Vanderlaan, M., Blank, G S., Performance comparison of protein A affinity-chromatography sorbents for purifying recombinant monoclonal antibodies. Biotechnol Appl Biochem 1999, 30, 121—128. 

Romaschin AD, Kirsten E. Jackowski G, Kun E (1981) Quantitative isolation of oligo- and polyadenosine-diphosphoribosylated proteins by affinity chromatography from livers of normal and dimethylnitrosamine-treated Syrian hamsters. J Biol Chem 256 7800-7805... 

Affinity chromatography (12) has become an important tool in the isolation of purified fractions of such substances as enzymes. Advantage is taken of specific interactions such as antigen-antibody interactions. One substance of the pair (e.g. antigen) is bonded to a support. When a mixture is passed through the column, the specific interaction retains the corresponding antibody relative to other substances. A change of mobile phase conditions then elutes the pure antibody. This method has a real potential for analysis of specific proteins in body fluids. 

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