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- Table of Contents
KMT2A / Histone-lysine N-methyltransferase 2A · Western blot design guide
Design a Western Blot for KMT2A
Real validated KMT2A Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-KMT2A 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
KMT2A 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 | ~431.8 kDa | |
| Gel | 4–12% gradient (standard starting point) | |
| Positive control | Placenta (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 | Cleavage-product bands | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated KMT2A Western Blot Protocols
The M00402 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Target-positive lysate and matched negative control (standard starting point) |
| Gel % | 4–12% gradient (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M00402; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected KMT2A Western Blot Band Size?
Full-length KMT2A is predicted at 431.8 kDa; cleavage and isoforms may affect bands, but native migration is not demonstrated by the supplied blot.
| Band near 431.8 kDa | consistent with predicted full-length KMT2A; identity requires confirmation |
| Bands below 431.8 kDa | may represent KMT2A cleavage products; fragment migration is not supplied |
| Several bands at different positions | could reflect cleavage or isoforms 1, 2, and 3; distinct isoform bands are not established |
| Little signal in a cytosolic fraction | KMT2A is nuclear |
💡Expected KMT2A appearanceFull-length KMT2A is predicted at 431.8 kDa; the supplied blot tests truncated constructs, so no native band position is established and band identity needs epitope-aware controls.
| Predicted full-length mass | 431.8 kDa is the sequence-based reference, not a measured band |
| Isoforms 1, 2, and 3 | may differ in size, but individual masses and migration are not supplied |
| KMT2A proteolytic cleavage | can yield bands below the full-length protein; fragment masses are not supplied |
| Phosphorylation at Ser136 and Ser142 | may affect mobility, but a visible shift is not established |
| Acetylation at Lys239 | may affect mobility, but a visible shift is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | nuclear KMT2A may be poorly recovered | check nuclear extraction and use a nuclear-enriched sample |
| Band lower than expected | KMT2A cleavage or a truncated construct | check the antibody epitope and compare antibodies against different regions |
| Multiple bands | cleavage products or isoforms 1, 2, and 3 | compare regional antibodies and confirm band identity with KMT2A depletion |
| Weak or no signal | poor recovery of nuclear KMT2A or an epitope absent from a cleavage product | check nuclear enrichment and the antibody epitope |
| Fragments below expected size | KMT2A cleavage products or sample degradation | compare promptly prepared samples and antibodies against different regions |
Experiment evidence
Western blot images showing KMT2A band
M00402 · KMT2A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for KMT2A Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for KMT2A in Western blot, you can use placenta lysate, which has high HPA expression.
Positive control: Placenta (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: No tissue is listed as not detected, so use siRNA knockdown or a KO line for a clean negative control.
HPA tissue expression evidence for KMT2A
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 |
|---|---|---|---|---|
| Placenta | endothelial cells | High | Protein (IHC) | HPA → |
| Testis | pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Caudate | glial cells | Low | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Low | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Low | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced KMT2A Western Blot Tips
Deeper troubleshooting and optimisation questions for KMT2A, answered from its protein features.
Where should KMT2A bands appear relative to its predicted mass?
Band shift · The predicted 431.8 kDa refers to full-length KMT2A. UniProt also describes N320 and C180 cleavage products, so a band below 431.8 kDa could represent a product. No observed band position is supplied; the listed modifications alone cannot establish a visible shift or explain a mass difference.
Could KMT2A isoforms produce different bands?
Isoforms · Three isoforms are listed. Isoform 2 lacks residues 1407–1444; isoform 3 has S replaced by SGTE at position 1603. These sequence differences may affect which forms an antibody recognizes, but the features do not establish that separate bands will resolve.
Which KMT2A modifications matter when interpreting bands?
PTM · UniProt lists 33 modified residues, including CK2 phosphorylation at Ser136 and Ser142, acetylated lysines, and autocatalytic S-methylcysteine at Cys3882. These are UniProt coordinates; antibody or paper numbering may differ. Modification presence does not demonstrate a detectable mobility shift.
Does this guide establish induction of KMT2A?
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 KMT2A Western blot?
Transfer · KMT2A is predicted at 431.8 kDa, and UniProt describes N320 and C180 cleavage products. Check transfer of the high-mass region and the regions targeted by your antibody. The supplied features do not specify a membrane, buffer, or transfer setting.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M00402 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 KMT2A Western blot signals be quantified?
Quantitation · Define whether the measurement targets full-length KMT2A, N320, or C180 before quantifying. Use an antibody whose recognized region matches that target, and quantify the same band across samples. KMT2A is listed as nuclear, so keep the sample fraction consistent.
How can KMT2A cleavage affect antibody band detection?
Interpretation · UniProt describes N320 and C180 cleavage products that heterodimerize. Check whether the antibody recognizes the N-terminal or C-terminal portion before assigning a band to either product. Their names do not establish exact Western blot migration positions.
Consider the described N320 and C180 cleavage products and the three isoforms when assessing extra bands. Compare each band with the antibody’s recognized region and the isoform sequence changes. The listed phosphorylation and acetylation sites do not, by themselves, identify an unexpected band.
Consider the described N320 and C180 cleavage products and the three isoforms when assessing extra bands. Compare each band with the antibody’s recognized region and the isoform sequence changes. The listed phosphorylation and acetylation sites do not, by themselves, identify an unexpected band.
Boster reagents
KMT2A 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-MLL KMT2A Monoclonal Antibody
Cat # M00402
M00402 is a human-reactive anti-KMT2A monoclonal antibody with a WB image showing truncated recombinant MLL and truncated GFP-MLL(aa3714-3969) in transfected Cos7 lysate. The supplied evidence does not show endogenous, full-length KMT2A.
Which to pick: M00402 is the only listed antibody and has a WB image. Choose it if its reported truncated-protein WB conditions suit your experiment; suitability for endogenous, full-length KMT2A needs separate assessment.
Source: BosterBio KMT2A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q03164.
- Human Protein Atlas. KMT2A tissue expression.
- PMC7288919 — target-verified WB comparison
- PMC6601586 — target-verified WB comparison
- PMC11782969 — target-verified WB comparison