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DNMT3A / DNA (cytosine-5)-methyltransferase 3A · Western blot design guide
Design a Western Blot for DNMT3A
Real validated DNMT3A Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-DNMT3A 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
DNMT3A 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 | ~101.9 kDa | |
| Observed band | ~120–130 kDa | |
| Gel | 10% (catalog A00136-2) | |
| Positive control | Adrenal gland (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 + Methylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated DNMT3A Western Blot Protocols
The A00136-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human MCF-7, rat testis (catalog A00136-2) |
| Gel % | 10% (catalog A00136-2) |
| Load | 30 ug; reducing conditions (catalog A00136-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00136-2) |
| Membrane | nitrocellulose membrane (catalog A00136-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00136-2) |
| Primary antibody | A00136-2 · 0.5 μg/ml (catalog A00136-2) |
| Primary incubation | overnight at 4°C (catalog A00136-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00136-2) |
| Secondary incubation | 1.5 hour at RT (catalog A00136-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00136-2) |
| Detection | ECL (catalog A00136-2) |
Section 2
What Is the Expected DNMT3A Western Blot Band Size?
DNMT3A is predicted at 101.9 kDa, while antibody blots report ~120–130 kDa; the cause of the difference is not established.
| Band at ~120–130 kDa | Empirical DNMT3A band reported in reducing whole-cell blots; confirm identity with appropriate controls. |
| Band near 102 kDa | Near the 101.9 kDa predicted mass; identity still requires confirmation. |
| Several bands at different positions | Isoforms 1, 2, and 3 are annotated, but their migration differences are unknown. |
| Higher band near twice the monomer size | Could reflect retained DNMT3A homodimer; no interchain disulfide is annotated. |
💡Expected DNMT3A appearanceDNMT3A has a predicted mass of 101.9 kDa, while reducing whole-cell blots report ~120–130 kDa; the cause of this difference is unestablished, so confirm band identity with ordinary controls.
| Predicted mass of 101.9 kDa | Provides the sequence-based reference; the empirical band is ~120–130 kDa for an unestablished reason. |
| Splice isoform 1 | Its apparent size relative to isoforms 2 and 3 is not supplied. |
| Splice isoform 2 | Its apparent size relative to isoforms 1 and 3 is not supplied. |
| Splice isoform 3 | Its apparent size relative to isoforms 1 and 2 is not supplied. |
| DNMT3A homodimer | Could produce a higher band if association survives electrophoresis; no interchain disulfide is annotated. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Chromatin-associated DNMT3A may be poorly recovered by the extraction method. | Check nuclear and chromatin extraction with a positive control. |
| Band higher than expected | The reported ~120–130 kDa band exceeds the 101.9 kDa prediction for an unestablished reason. | Compare with a validated positive control and confirm antibody specificity. |
| Band lower than expected | An alternative isoform is possible, but its size is not supplied. | Check antibody epitope coverage and compare with a validated control. |
| Multiple bands | Isoforms 1, 2, and 3 are annotated, though distinct migration is unproven. | Compare isoform-aware controls and assess antibody specificity. |
| Weak or no signal | Nuclear or chromatin-associated protein may be underrepresented in the prepared sample. | Assess extraction and loading with suitable controls. |
Experiment evidence
Western blot images showing DNMT3A band
A00136-2 · DNMT3A
A00136-2 · DNMT3A
M00136 · DNMT3A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for DNMT3A Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for DNMT3A in Western blot, you can use adrenal gland lysate, which shows high HPA expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside samples.
⚠️Feasibility: No tissue is listed as not detected, so use siRNA knockdown or a KO line for a negative control.
HPA tissue expression evidence for DNMT3A
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Breast | adipocytes | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Medium | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Medium | Protein (IHC) | HPA → |
Section 3
Advanced DNMT3A Western Blot Tips
Deeper troubleshooting and optimisation questions for DNMT3A, answered from its protein features.
How should DNMT3A band migration be interpreted?
Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.
How could isoform 2 affect the band pattern?
Isoforms · Isoform 2 replaces canonical UniProt residues 1–213 with a shorter sequence. An antibody targeting that replaced region may miss isoform 2. Check the antibody epitope before assigning a smaller band to this isoform.
Isoform 3 replaces canonical UniProt residues 151–166 and lacks residues 167–912. It could therefore produce a much smaller band if the antibody recognizes a retained region. An antibody targeting residues 167–912 would not detect it.
Isoform 3 replaces canonical UniProt residues 151–166 and lacks residues 167–912. It could therefore produce a much smaller band if the antibody recognizes a retained region. An antibody targeting residues 167–912 would not detect it.
Which DNMT3A modifications matter when interpreting bands?
PTM · In canonical UniProt numbering, phosphorylation is listed at Ser105, Thr124, Ser243, Ser255, Thr261, Ser267, Ser390 and Ser393; omega-N-methylarginine at Arg171; and autocatalytic S-methylcysteine at Cys710. These sites may guide follow-up experiments, but the feature list does not show that any modification causes a visible shift.
Does this guide establish induction of DNMT3A?
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 DNMT3A Western blot?
Transfer · DNMT3A has a predicted mass of 101.9 kDa, and an apparent band has been reported around 120–130 kDa. Check transfer efficiency across that size range with a molecular weight marker. The supplied features do not identify a specific transfer method as optimal.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00136-2 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 I quantify DNMT3A bands across samples?
Quantitation · Define which band and isoforms your antibody detects, then quantify the same band consistently across samples. DNMT3A is listed in the nucleus, on chromosomes and in the cytoplasm, so use comparable sample fractions when interpreting changes.
Why might DNMT3A appear at 120–130 kDa?
Interpretation · The canonical DNMT3A sequence predicts 101.9 kDa, while the reported Western blot band is approximately 120–130 kDa. UniProt lists isoforms and modifications, but their presence alone does not establish the cause of this difference. Compare the band with a molecular weight marker and check whether the antibody detects the canonical sequence.
Check its size against the 101.9 kDa canonical prediction and the reported 120–130 kDa band. Consider isoform 2’s shorter N-terminal replacement and isoform 3’s loss of canonical residues 167–912, then check whether the antibody recognizes the relevant sequence. Listed modifications alone cannot identify an unexpected band.
Check its size against the 101.9 kDa canonical prediction and the reported 120–130 kDa band. Consider isoform 2’s shorter N-terminal replacement and isoform 3’s loss of canonical residues 167–912, then check whether the antibody recognizes the relevant sequence. Listed modifications alone cannot identify an unexpected band.
Boster reagents
DNMT3A 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-DNMT3A Antibody Picoband®
Cat # A00136-2
Real WB data
Anti-Dnmt3a Rabbit Monoclonal Antibody
Cat # M00136
Two the supplier anti-DNMT3A antibodies are listed for Western blotting: polyclonal A00136-2 and monoclonal M00136. Both list human, mouse, and rat reactivity and have blot images with named samples. No independent specificity validation is supplied.
Which to pick: Choose by sample context: A00136-2 has images using 293T, MCF-7, rat and mouse testis, and NIH/3T3 lysates; M00136 uses SH-SY5Y, MCF-7, rat and mouse brain, and mouse lung. Their reported bands are approximately 120–130 kDa and 130 kDa, respectively.
Source: BosterBio DNMT3A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9Y6K1.
- Human Protein Atlas. DNMT3A tissue expression.
- PMC9615155 — target-verified WB comparison
- PMC11725086 — target-verified WB comparison
- PMC11739694 — target-verified WB comparison