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Quantitative Immunofluorescence to Measure Global Localized Translation
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Fluorescent foci quantitation for high-throughput analysis.

Elena Ledesma-Fernández1, Peter H Thorpe1

  • 1The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London, United Kingdom NW71AA.

Journal of Biological Methods
|August 21, 2015
PubMed
Summary
This summary is machine-generated.

Researchers developed FociQuant, a free tool for high-throughput quantitation of fluorescent protein foci. This tool aids in studying kinetochore function and protein levels, crucial for understanding cell division and cancer.

Keywords:
ImageJcerevisiaefluorescencekinetochoreyeast

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Area of Science:

  • Cell Biology
  • Genetics
  • Biophysics

Background:

  • Cellular proteins often form discrete foci, such as kinetochores, essential for chromosome segregation.
  • Measuring fluorescence in these foci can quantify protein concentration, vital for cell division and cancer research.
  • Accurate chromosome segregation relies on kinetochore function; perturbations lead to aneuploidy, a cancer hallmark.

Purpose of the Study:

  • To develop freely-available tools for high-throughput quantitation of fluorescence foci intensities.
  • To establish a proof-of-principle for quantifying kinetochore protein levels using fluorescence.
  • To enable analysis of any protein forming discrete cellular foci.

Main Methods:

  • Creation of a novel software tool, 'FociQuant', for automated fluorescence foci quantitation.
  • Application of FociQuant to budding yeast kinetochores, which form a single nuclear focus.
  • Comparison of different kinetochore quantitation methods using FociQuant.

Main Results:

  • FociQuant enables high-throughput quantitation of fluorescence foci.
  • The tool successfully quantifies changes in kinetochore protein levels in a functional mutant.
  • Proof-of-principle demonstrated for analyzing kinetochore protein homeostasis.

Conclusions:

  • FociQuant provides a valuable tool for high-throughput analysis of protein foci.
  • This method can be applied to study kinetochore regulation and other protein foci in cell biology.
  • Understanding protein foci dynamics is crucial for cell division, genome integrity, and cancer research.