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Updated: Dec 11, 2025
Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks
Published on: November 3, 2017
Detection of DNA Double-Strand Breaks by γ-H2AX Immunodetection
Sonia I Barroso1, Andrés Aguilera2
1Centro Andaluz de Biología Molecular y Medicina Regenerativa, CABIMER, University of Seville-CSIC-UPO, Seville, Spain.
Abstract:
DNA double-strand breaks (DSBs) are the most deleterious type of DNA damage and a cause of genetic instability as they can lead to mutations, genome rearrangements, or loss of genetic material when not properly repaired. Eukaryotes from budding yeast to mammalian cells respond to the formation of DSBs with the immediate phosphorylation of a histone H2A isoform. The modified histone, phosphorylated in serine 139 in mammals (S129 in yeast), is named γ-H2AX. Detection of DSBs is of high relevance in research on DNA repair, aging, tumorigenesis, and cancer drug development, given the tight association of DSBs with different diseases and its potential to kill cells. DSB levels can be obtained by measuring levels of γ-H2AX in extracts of cell populations or by counting foci in individual nuclei. In this chapter some techniques to detect γ-H2AX are described.
Insights
DNA double-strand breaks (DSBs) are dangerous DNA damage. Detecting the marker gamma-H2AX helps study DNA repair, aging, and cancer development.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- DNA double-strand breaks (DSBs) represent the most severe form of DNA damage.
- Unrepaired DSBs lead to genetic instability, mutations, and genomic rearrangements.
- DSBs are implicated in aging, tumorigenesis, and cancer progression.
Purpose of the Study:
- To describe techniques for detecting gamma-H2AX, a marker for DSBs.
- To highlight the relevance of DSB detection in various research fields.
Main Methods:
- Detection of DSBs by measuring gamma-H2AX levels in cell extracts.
- Quantification of DSBs by counting gamma-H2AX foci in individual cell nuclei.
Main Results:
- Gamma-H2AX is a reliable biomarker for DSBs across eukaryotes.
- Specific techniques for gamma-H2AX detection are detailed in the chapter.
Conclusions:
- Accurate detection of DSBs is crucial for understanding DNA repair mechanisms.
- Gamma-H2AX detection is vital for research in aging, cancer, and drug development.
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02:31Homologous Recombination