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1D4: A Versatile Epitope Tag for the Purification and Characterization of Expressed Membrane and Soluble Proteins

  • Laurie L. Molday
  • Robert S. MoldayEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1177)

Abstract

Incorporation of short epitope tags into proteins for recognition by commercially available monoclonal or polyclonal antibodies has greatly facilitated the detection, characterization, localization, and purification of heterologously expressed proteins for structure–function studies. A number of tags have been developed, but many epitope–antibody combinations do not work effectively for all immunochemical techniques due to the nature of the tag and the specificity of the antibodies. A highly versatile, multipurpose epitope tag is the 9 amino acid C-terminal 1D4 peptide. This peptide tag together with the Rho1D4 monoclonal antibody can be used to detect proteins in complex mixtures by western blotting and ELISA assays, localize proteins in cells by immunofluorescence and immunoelectron microscopic labeling techniques, identify subunits and interacting proteins by co-immunoprecipitation, and purify functionally active proteins including membrane proteins by immunoaffinity chromatography. In this chapter we describe various immunochemical procedures which can be used for the detection, purification and localization of 1D4-tagged proteins for structure–function studies.

Key words

1D4 epitope tag Rho1D4 antibody 1D4-tagged proteins Immunoaffinity chromatography Immunocytochemical localization Co-immunoprecipitation Membrane protein purification Protein expression 

Notes

Acknowledgments

This work was supported by grants from the National Institutes of Health (EY002422-32) and the Canadian Institutes of Health Research (CIHR MOP-106667). RSM is a Canada Research Chair in Vision and Macular Degeneration.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of British ColumbiaVancouverCanada

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