Immunofluorescence analysis of endogenous and exogenous centromerekinetochore proteins

Yohei Niikura, Katsumi Kitagawa

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

“Centromeres” and “kinetochores” refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins. 1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromerekinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Original languageEnglish (US)
Article numbere53732
JournalJournal of Visualized Experiments
Volume2016
Issue number109
DOIs
StatePublished - Mar 3 2016
Externally publishedYes

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Keywords

  • Cellular biology
  • CENP-A
  • Centromere
  • Indirect immunofluorescent staining (IIF)
  • Issue 109
  • Kinetochore
  • Mitosis
  • Post-translational modification (PTM)
  • Western blot (WB)

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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