Abstract
Affinity chromatography relies on the reversible interaction between a protein and a specific ligand immobilized in a chromatographic matrix. The sample is applied under conditions that favor specific binding to the ligand as the result of electrostatic and hydrophobic interactions, van der Waals’ forces and/or hydrogen bonding. After washing away the unbound material the bound protein is recovered by changing the buffer conditions to those that favor desorption. The technique has been used not only to isolate antigen-specific antibodies but also to remove specific contaminants from biological samples. Methods are described for the purification of immunoglobulins, namely IgG, IgG fragments and subclasses, using the high affinity of protein A and protein G coupled to agarose. In the Subheading 3 there are also protocols for affinity purification using a specific ligand coupled to commercial matrices like CNBr- Sepharose 4-B and Affigel.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Protein purification handbook. GE Health Care 800 Centennial Avenue, Piscataway, NJ08855. (http://www1.gelifesciences.com/aptrix/upp00919.nsf/Content/9C7BA3 DA6539F07AC1256EB40044A8B2/$file/18113229AC.pdf)
Affinity chromatography handbook. Principles and methods. Amersham Biosciences. (http://www1.gelifesciences.com/aptrix/upp00919.nsf/Content/91D3DF5DE303E8B6C1256EB400417F34/$file/18102229AD.pdf)
Antibody. production, purification, fragmentation, labeling. Pierce Biotechnology. P.O. Box 117, Rockford, IL61105. Part 1: http://www.piercenet.com/files/1601323%20Antibodies_1.pdf; Part 2: http://www.piercenet.com/files/1601323%20Antibodies_2.pdf; Part 3: http://www.piercenet.com/files/1601323%20Antibodies_3.pdf
Bauer K, Bayer PM, Deutsch E, Gabl F (1980) Binding of enzyme-IgG complexes in human serum to Protein-A Sepharose CL-4B. Clin Chem 26:297–300
Protein A agarose. Invitrogen. Carlsbad, CA. (https://www.invitrogen.com/content/sfs/manuals/15918014.pdf)
Acknowledgement
This work was supported by the Intramural Research Program of the NIH, NIDCR.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Grodzki, A.C., Berenstein, E. (2010). Antibody Purification: Affinity Chromatography – Protein A and Protein G Sepharose. In: Oliver, C., Jamur, M. (eds) Immunocytochemical Methods and Protocols. Methods in Molecular Biology, vol 588. Humana Press. https://doi.org/10.1007/978-1-59745-324-0_5
Download citation
DOI: https://doi.org/10.1007/978-1-59745-324-0_5
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-463-0
Online ISBN: 978-1-59745-324-0
eBook Packages: Springer Protocols