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Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes
Published on: December 20, 2014
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Protocol to measure inter-centrosome distance in adherent cells using epifluorescence microscopy
Franco K C Au1, Edna S W Lui1, Robert Z Qi1,2
1Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China.
STAR Protocols
|March 14, 2022
Summary
This study details a method to measure centrosome separation during the cell cycle using immunofluorescence microscopy. The protocol allows researchers to investigate proteins involved in centrosome cohesion and separation in various cell types.
Area of Science:
- Cell Biology
- Molecular Biology
- Microscopy
Background:
- Centrosome separation is crucial for cell division.
- Understanding the molecular mechanisms regulating centrosome separation is essential for cell cycle research.
- Existing methods may not fully capture dynamic centrosome separation events.
Purpose of the Study:
- To present a detailed protocol for assaying centrosome separation at the late-G2 phase of the cell cycle.
- To enable the imaging and measurement of inter-centrosome distance.
- To provide a versatile method for testing proteins involved in centrosome separation and cohesion.
Main Methods:
- Immunofluorescence microscopy to visualize centrosomes.
- Detailed steps for imaging and quantifying inter-centrosome distance.
- Application in hTERT RPE-1 cells, adaptable to other adherent cell lines like U2OS and MRC-5.
Main Results:
- A robust protocol for assessing centrosome separation is established.
- The method allows for quantitative measurement of inter-centrosome distances.
- GAS2L1 is presented as an example protein for investigation.
Conclusions:
- The presented protocol offers a reliable method to study centrosome separation dynamics.
- This technique can be broadly applied to investigate the function of various proteins in centrosome cohesion and separation.
- The protocol is suitable for different adherent cell lines, enhancing its utility in cell cycle research.
