Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.prot4557

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Using FLAG Epitope-Tagged Proteins for Coimmunoprecipitation of Interacting Proteins

Anne Verhagen

This protocol was adapted from "Peptide Mapping and Sequence Analysis of Gel-Resolved Proteins," Chapter 10 in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.


INTRODUCTION

This protocol describes the use of FLAG-epitope-tagged proteins for both small-scale analysis and large-scale coimmunoprecipitation of interacting proteins. When examining protein interactions, it is sometimes possible to immunoprecipitate an endogenous protein X directly, without using an epitope tag, if antibodies are available. The advantage of examining interactions of endogenously expressed proteins is that these are more likely to be physiological and less likely to be an artifact of overexpression. However, when scaling up the procedure for protein isolation and identification, epitope-tagged overexpressed protein X (e.g., FLAG-protein X) may be necessary for sufficient coimmunoprecipitating protein to be isolated.


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