SpringerProtocols: Abstract: 2. Purification and Assay of Kinase-Active EGF Receptor From Mammalian Cells by Immunoaffinity Chromatography

2. Purification and Assay of Kinase-Active EGF Receptor From Mammalian Cells by Immunoaffinity Chromatography

By: Gregory J. Wiepz³, Arturo G. Guadaramma⁴, David L. Fulgham⁵, Paul J. Bertics⁶

Abstract

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The epidermal growth factor (EGF) receptor possesses intrinsic protein-tyrosine kinase activity, and both overexpressed wild-type and mutated forms have been associated with many types of cancers. Therefore, understanding the mechanisms that modulate receptor activity and function is essential to the development of treatments for many of these cancers. However, to address this issue by either conventional or high-throughput screening methods requires the availability of large amounts of highly purified and active EGF receptor.

The technique described in this chapter utilizes immunoaffinity chromatography, which allows for the isolation of highly purified and active preparations of EGF receptor. By immobilizing an antibody that recognizes the ligand-binding domain of the receptor to Sepharose beads, the receptor can be eluted specifically from the antibody by the addition of EGF. This association establishes a unique interaction that ensures the isolation of a highly enriched preparation of EGF receptor. This protocol allows for the purification of large or small batches of receptor that retain their kinase activity. Additionally, this chapter reports on the subsequent steps necessary to characterize the receptor: kinase activity, mass, purity, and the ability of the receptor to undergo autophosphorylation.

Affiliation(s): (3) Department of Biomolecular Chemistry, University of Wisconsin, WI
(4) Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI
(5) Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI
(6) Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI

Book Title: Epidermal Growth Factor: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 327 | Pub. Date: Dec-15-2005 | Page Range: 25-38 | DOI: 10.1385/1-59745-012-X:25

Subject: Cell Biology

Key Words: Epidermal growth factor (EGF) receptor - tyrosine kinase - autophosphorylation - immunoaffinity chromatography - receptor purification

References

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