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PIN-G Reporter for Imaging and Defining Trafficking Signals in Membrane Proteins

  • Lynn Mckeown
  • Vicky C. Jones
  • Owen T. Jones
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 574)

Abstract

The identification of motifs that control the intracellular trafficking of proteins is a fundamental objective of cell biology. Once identified, such regions should, in principle, be both necessary and sufficient to direct any randomly distributed protein, acting as a reporter, to the subcellular compartment in question. However, most reporter proteins have limited versatility owing to their endogenous expression and limited modes of detection – especially in live cells. To surmount such limitations, we engineered a plasmid – pIN-G – encoding an entirely artificial, type I transmembrane reporter protein (PIN-G), containing HA, cMyc and GFP epitope, and fluorescence tags. Although originally designed for trafficking studies, pIN technology is a powerful tool applicable to almost every area of biology. Here we describe the methodologies used routinely in analyzing pIN constructs and some of their derivatives.

Key words

Green fluorescent protein (GFP) live imaging trafficking epitope tags reporter constructs photoactivation lentivirus 

Notes

Acknowledgments

This work was supported by funds from the Biotechnology and Biological Sciences Research Council UK: BBSRC, BB/D008891/1. We are indebted to Professor P.-L. Nicotera and Dr. D. Bano (University of Leicester, UK) for their gift of the lentiviral packaging system and advice on its use. We also thank Jane Kott and the University of Manchester Bioimaging Facility for imaging support.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Lynn Mckeown
    • 1
  • Vicky C. Jones
    • 1
  • Owen T. Jones
    • 1
  1. 1.Faculty of Life Sciences, The University of ManchesterManchesterUK

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