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Gamma-H2AX (also written γ-H2AX, gamma H2AX, gammaH2AX or phospho-Ser139 H2AX) is the form of histone H2AX that has been phosphorylated on serine 139. Cells make it within minutes of a DNA double-strand break, and each visible nuclear focus of it is generally read as one break. This page is general reference for researchers and lab staff, not a protocol or advice about any individual.
Short answer
Gamma-H2AX is phosphorylated histone H2AX. It was first described by Rogakou and colleagues in 1999 as the specific phosphorylation of H2AX on serine 139, and Mah et al. (2011) call it a sensitive and reliable marker of DNA double-strand breaks (DSBs). A focus count is a strong readout of breaks, but timing, dose, cell type and cell death all change how the number should be read.
Definition and scope
H2AX is a variant of histone H2A. “Gamma-H2AX” is not a separate protein: it names the phosphorylated state of H2AX. That distinction matters when you read a methods section or buy reagents, because an antibody against total H2AX and an antibody against the phosphorylated form answer different questions. The notation varies between papers (gamma H2AX, gammaH2AX, γ-H2AX, H2AX phospho-Ser139), and all of these refer to the same modification. The shorter term “H2AX” alone refers to the histone, whether or not it is phosphorylated.
In the original report, Rogakou et al. (NCI/NIH, Journal of Cell Biology 1999) described specific phosphorylation of H2AX on serine 139 and detected it with an antibody raised against the phosphorylated C-terminal peptide, using immunoblotting and confocal immunofluorescence. Mah et al. (Genome Integrity 2011) describe the phosphorylation as occurring at a conserved SQ motif and name ATM as one of the PI3K-related kinases that acts on it.
How it is used in practice
In the studies cited here, gamma-H2AX is measured with phospho-specific antibodies. Mah et al. describe immunofluorescence microscopy as the preferred method of DSB detection, with discrete nuclear foci that can be counted per cell; immunoblotting gives a bulk signal for a population instead of a per-cell count. Rogakou et al. also showed that breaks made locally with a 390 nm laser microbeam were used to create breaks in cells in order to study where the marker appears.
Two findings from these papers explain why the marker is popular. First, it appears fast: Rogakou et al. report that H2AX phosphorylation (measured in bulk) appeared within 1 minute of ionizing radiation in muntjac mitotic cells, was half-maximal at about 1 to 3 minutes and was maximal at about 10 to 30 minutes. In IMR90 cells, foci were first scored at 3 minutes, were most numerous at 3 minutes and had fallen to about 4.8 per nucleus by 180 minutes. Second, it scales with damage: the number of foci was comparable to the number of breaks induced, which led the authors to propose that each focus may represent one DSB in vivo.
What a gamma-H2AX focus does and does not tell you
The checklist below is built only from the two papers cited on this page. It is an interpretation aid for reading published data, not a validated assay procedure.
| Question to ask of a result | What the sources report | Source |
|---|---|---|
| When was the sample taken? | Bulk H2AX phosphorylation appears within 1 minute and is maximal at about 10 to 30 minutes (Rogakou 1999); in IMR90 cells foci were highest at the first scored time (3 minutes) and fell by 180 minutes; the reviewed radiation studies describe a peak at about 30 to 60 minutes; back to background within about 24 hours at lower doses. A single time point can miss the peak or the repair. | Rogakou 1999; Mah 2011 |
| How high was the dose? | Scoring individual foci is described as most accurate at doses below 4 Gy. | Mah 2011 |
| What is the baseline for this cell type? | Untreated baseline foci differ by cell type: roughly 1 to 2 per nucleus in normal cells, about 5 to 20 in cancer cell lines and over 100 in embryonic stem cells. | Mah 2011 |
| Could the cells be dying? | Apoptotic cells express extremely high gamma-H2AX, so a bulk method such as Western blotting cannot separate a repair response from apoptosis. | Mah 2011 |
| Where in the genome is the damage? | Foci are reported to form rarely in heterochromatin, so absence of a focus is not proof of absence of a break there. | Mah 2011 |
Related concepts and notation
H2AX versus gamma-H2AX
H2AX is the histone; gamma-H2AX is the Ser139-phosphorylated form of it. Total-H2AX staining shows where the histone is, not where damage is.
Foci versus bulk signal
Counting foci in single cells (immunofluorescence) reports breaks per nucleus; bulk methods report an average signal. The two can disagree when a minority of cells is apoptotic.
DNA damage and genome editing
Nuclease-based editing tools create double-strand breaks as part of their mechanism. For background on those tools see the CASRAI guides on zinc-finger nucleases, gene knockout and sgRNA design, and on base editing, which is designed to avoid a double-strand cut. How gamma-H2AX is used to monitor any specific editing experiment is a question for the study’s own methods.
Where it matters for labs
For lab managers and reviewers the practical point is documentation. Because the readout depends on time after exposure, dose, cell type and cell health, an SOP or methods section that reports a gamma-H2AX result should state these alongside the number so another lab can interpret it. Recording antibody identity, the counting method and the time point is the minimum needed to compare results; see also the CASRAI guide to DNA extraction protocol basics for the neighbouring bench context.
Common misunderstandings
- “Gamma-H2AX is the same as the H2AX protein.” It is one modified state of it.
- “Every focus is a break and every break is a focus.” The 1999 authors proposed that a focus may represent one break; the later review notes that foci rarely form in heterochromatin and that apoptosis raises the signal independently.
- “A higher signal always means more repair activity.” Apoptotic cells express extremely high gamma-H2AX, so a high bulk signal can reflect cell death.
- “The count is comparable across any dose and time.” Counting is described as most accurate below 4 Gy and the signal returns to background over hours.
FAQ
What does gamma-H2AX stand for?
It is histone H2AX phosphorylated on serine 139. The “gamma” prefix was used for the phosphorylated form in the 1999 report.
What is H2AX?
A variant of histone H2A. The unmodified histone is called H2AX; the phosphorylated form is gamma-H2AX.
Which kinase makes gamma-H2AX?
Mah et al. (2011) name ATM as a PI3K-related kinase that phosphorylates H2AX at its conserved SQ motif.
How fast does it appear and how long does it last?
Rogakou 1999 reports bulk H2AX phosphorylation within 1 minute of radiation, half-maximal at about 1 to 3 minutes and maximal at about 10 to 30 minutes; in IMR90 cells, foci were first scored at 3 minutes and had fallen by 180 minutes. Mah 2011 describes a peak at about 30 to 60 minutes and a usual return to background within about 24 hours at lower doses.
Sources
- Rogakou EP, Boon C, Redon C, Bonner WM. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 1999;146(5):905-916. PMC2169482. Read 2026-11-08.
- Mah LJ, Orlowski C, Ververis K, Vasireddy RS, El-Osta A, Karagiannis TC. Evaluation of the efficacy of radiation-modifying compounds using gammaH2AX as a molecular marker of DNA double-strand breaks. Genome Integrity 2011;2:3. doi:10.1186/2041-9414-2-3. PMC3037297. Read 2026-11-08.
