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Estimation of GFP-Nucleoporin Amount Based on Fluorescence Microscopy

  • Haruhiko Asakawa
  • Yasushi Hiraoka
  • Tokuko HaraguchiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1721)

Abstract

Cellular structures and biomolecular complexes are not simply assemblies of proteins, but are organized with defined numbers of protein molecules in precise locations. Thus, evaluating the spatial localization and numbers of protein molecules is of fundamental importance in understanding cellular structures and functions. The amounts of proteins of interest have conventionally been determined by biochemical methods. However, biochemical measurements based on the population average have limitations: it is sometimes difficult to determine the amounts of insoluble proteins or low expression proteins localized in small portions of the cell. In contrast, microphotometric measurements using fluorescence microscopes enable us to detect the amounts of such proteins in situ in a particular subcellular region. Here, we describe a method to measure the amounts of fluorescently tagged proteins by fluorescence microscopy, and present an example of an application to nuclear pore proteins in the fission yeast Schizosaccharomyces pombe.

Key words

Live cell imaging Fluorescence microscopy Microphotometry Nuclear pore complex Nuclear structures 

Notes

Acknowledgments

This work was supported by JSPS Kakenhi Grant Numbers, JP26440098 to H.A., JP16H01309 to Y.H., and JP25116006 to T.H.

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Haruhiko Asakawa
    • 1
  • Yasushi Hiraoka
    • 1
    • 2
  • Tokuko Haraguchi
    • 1
    • 2
    Email author
  1. 1.Graduate School of Frontier BiosciencesOsaka UniversitySuitaJapan
  2. 2.Advanced ICT Research Institute KobeNational Institute of Information and Communications TechnologyKobeJapan

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