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- Table of Contents
H2AX / Histone H2AX · Western blot design guide
Design a Western Blot for H2AX
Real validated H2AX Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-H2AX WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use
H2AX Western Blot Experimental Design Guide
Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~15.1 kDa | |
| Observed band | ~17 kDa | |
| Gel | 10% (catalog A00241-4) | |
| Positive control | Bone marrow (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated H2AX Western Blot Protocols
The A00241-4 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human 293T, human Raji (catalog A00241-4) |
| Gel % | 10% (catalog A00241-4) |
| Load | 30 ug; reducing conditions (catalog A00241-4) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00241-4) |
| Membrane | nitrocellulose membrane (catalog A00241-4) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00241-4) |
| Primary antibody | A00241-4 · 1:1000 (catalog A00241-4) |
| Primary incubation | overnight at 4°C (catalog A00241-4) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00241-4) |
| Secondary incubation | 1.5 hour at RT (catalog A00241-4) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00241-4) |
| Detection | ECL (catalog A00241-4) |
Section 2
What Is the Expected H2AX Western Blot Band Size?
The selected total-H2AX antibody shows a band at approximately 17 kDa. Its caption gives 15 kDa as the expected band size, while the reviewed UniProt sequence has a calculated mass of 15.145 kDa. The mass difference does not establish a cause or a distinct modification-related shift.
| Band near 17 kDa | Matches the band observed with total-H2AX antibody A00241-4 across the lysates shown in the selected lead. Confirm identity with appropriate controls. |
| Band near 17 kDa after nuclear enrichment | Consistent with the observed lead band and H2AX's nuclear and chromosomal location; compare with a validated H2AX control. |
| A closely spaced band near 17 kDa | A modified state is possible, but the supplied evidence does not establish visible separation or identify the modification. |
| Weak or absent band in a whole-cell lysate | Chromatin-associated H2AX may be underrepresented after sample preparation; check recovery and loading. |
💡Expected H2AX appearanceThe selected total-H2AX lead shows a band at approximately 17 kDa, versus its caption's expected 15 kDa and UniProt's calculated 15.145 kDa. Listed modifications do not establish the cause of that difference or a detectable shift. Confirm band identity with controls; use a phospho-specific reagent to assess phosphorylation.
| Calculated and observed molecular weight | UniProt calculates 15.145 kDa; the lead caption states an expected 15 kDa and observes approximately 17 kDa with total-H2AX antibody A00241-4. The difference has no established cause. |
| Ser-140 phosphorylation | UniProt lists phosphorylation by ATM, ATR and PRKDC. Gamma-H2AX literature commonly calls the C-terminal site Ser139; distinguish that convention from the supplied UniProt Ser140 coordinate and verify sequence and reagent mapping. Neither annotation establishes a visible shift. |
| Other phosphorylated sites | UniProt lists phosphoserine at Ser2 and Ser122 and phosphotyrosine at Tyr143 by WSTF. These annotations do not establish separate bands. |
| Acetylation and alternate lactoylation | UniProt lists N-acetylserine at Ser2, acetyllysine at Lys6, Lys10 and Lys37, and alternate lactoylation at Lys10. These annotations do not establish band separation. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Chromatin-associated H2AX may be poorly recovered | Check nuclear or chromatin extraction and use an H2AX positive control |
| Band higher than expected | The selected lead observes approximately 17 kDa despite an expected 15 kDa; the cause of the difference is unverified | Compare with the validated H2AX control and check antibody specificity |
| Band lower than expected | Possible degradation or unrelated antibody signal | Use protease inhibitors and compare with a validated H2AX control |
| Multiple bands | Modified states or nonspecific binding are possible; the supplied annotations do not establish distinct bands | Check antibody specificity and compare with a validated H2AX control; use site-specific assays to assess a proposed modification |
| Weak or no signal | Poor histone recovery or insufficient sample loading | Check histone extraction, loading and transfer |
| Fragments below expected size | Possible proteolysis during preparation | Use protease inhibitors and prepare a fresh sample |
Experiment evidence
Western blot images showing H2AX band
A00241-4 · H2AX
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for H2AX Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for H2AX in Western blot, you can use bone marrow lysate, which has high HPA expression.
Positive control: Bone marrow (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: H2AX is nuclear and detected across the listed tissues, so use a knockdown or KO line for a clear negative control.
HPA tissue expression evidence for H2AX
Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Higher expression tissues · candidate positive controls from IHC
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Caudate | glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | adipocytes | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced H2AX Western Blot Tips
Deeper troubleshooting and optimisation questions for H2AX, answered from its protein features.
Where should the main H2AX band appear?
Band shift · The selected total-H2AX antibody A00241-4 detects a band at approximately 17 kDa. Its caption states an expected 15 kDa, and reviewed UniProt calculates 15.145 kDa. Use the observed position as a reference for this lead and confirm band identity with controls; the source does not explain the difference.
Are multiple H2AX bands explained by isoforms?
Isoforms · The supplied record lists one isoform and no alternate isoform names. Do not assign additional bands to H2AX isoforms on this evidence; assess other explanations, including modification and antibody specificity.
Could H2AX modifications affect band interpretation?
PTM · Yes, but the annotations do not prove a visible shift. UniProt lists N-acetylserine and phosphoserine at Ser2; acetyllysine at Lys6, Lys10 and Lys37; alternate lactoylation at Lys10; phosphoserine at Ser122 and Ser140; and phosphotyrosine at Tyr143. Ser140 is annotated with ATM, ATR and PRKDC, and Tyr143 with WSTF. Band position alone cannot identify a modification or explain the observed 17 kDa position.
Does this guide establish induction of H2AX?
Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.
What transfer method to use for H2AX Western blot?
Transfer · Check retention of this small protein during transfer. The selected lead used a nitrocellulose membrane at 150 mA for 50–90 minutes and observed a band at approximately 17 kDa; verify that these conditions suit your setup.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00241-4 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.
How should H2AX signals be quantified?
Quantitation · Define whether the measurement is total H2AX or a specific modified form. A00241-4 is the total-target reagent in the selected lead; a phospho-H2AX measurement requires a phospho-specific reagent with verified site and numbering. Use a consistently defined signal across samples. The supplied evidence provides no expected fold change.
When is a DNA-damage comparison relevant for H2AX?
Interpretation · H2AX participates in the DNA-damage response. UniProt lists C-terminal Ser140 phosphorylation by ATM, ATR and PRKDC. Gamma-H2AX literature and some antibodies commonly call the C-terminal site Ser139; check the reagent's sequence and numbering convention before equating those labels. Use a phospho-specific reagent to measure phosphorylation under DNA-damage conditions; the selected A00241-4 lead is a total-H2AX assay and does not establish a phospho-specific signal or size change.
H2AX is listed in the nucleus and chromosome and is part of the nucleosome core. Assess nuclear or chromatin-associated fractions and consider recovery when interpreting a weak signal elsewhere.
H2AX is listed in the nucleus and chromosome and is part of the nucleosome core. Assess nuclear or chromatin-associated fractions and consider recovery when interpreting a weak signal elsewhere.
Boster reagents
H2AX Western Blot Antibodies
Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.
Real WB data
Anti-Histone H2A.X/H2AFX Antibody
Cat # A00241-4
The catalog reports one anti-H2AX antibody, A00241-4, with a Western blot analysis image for Histone H2A.X/H2AFX. The image caption names human HeLa, 293T, and Raji whole cell lysates; monkey COS-7 whole cell lysate; rat kidney tissue and C6 whole cell lysates; and mouse kidney tissue and 3T3-L1 whole cell lysates. These examples do not establish broader validation.
Which to pick: A00241-4 is the only listed option; its Western blot image covers the named human, monkey, rat, and mouse samples.
Source: BosterBio H2AX gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P16104.
- Human Protein Atlas. H2AX tissue expression.
- PMC6297734 — target-verified WB comparison
- PMC4385844 — target-verified WB comparison
- PMC12026332 — target-verified WB comparison