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- Table of Contents
DOT1L / Histone-lysine N-methyltransferase, H3 lysine-79 specific · Western blot design guide
Design a Western Blot for DOT1L
Real validated DOT1L Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-DOT1L 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
DOT1L 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 | ~164.9 kDa | |
| Gel | 8–10% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated DOT1L Western Blot Protocols
The M00840 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | RAW264.7 cell lysate (catalog M00840) |
| Gel % | 8–10% (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 | M00840; 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 DOT1L Western Blot Band Size?
DOT1L is predicted at 164.9 kDa; isoforms and phosphorylation could affect migration, but no empirical band or visible shift is established.
| Band near 164.9 kDa | consistent with the UniProt predicted mass; confirm identity with controls |
| Two bands at different positions | could reflect isoforms 1 and 2; their migration is not supplied |
| Closely spaced doublet near 164.9 kDa | could reflect isoforms or phosphorylation; neither visible pattern is established |
| No band in whole-cell lysate | nuclear DOT1L may require adequate nuclear extraction |
💡Expected DOT1L appearanceUniProt predicts 164.9 kDa for DOT1L; no empirical band size is supplied, and isoform or phosphorylation effects on migration are unverified, so confirm any candidate band with identity controls.
| UniProt predicted molecular weight | places the reference size at 164.9 kDa |
| Full-length 1537-residue sequence | underlies the predicted mass; no cleavage feature is listed |
| Splice isoform 1 | may differ in size from isoform 2; its individual mass is not supplied |
| Splice isoform 2 | may differ in size from isoform 1; its individual mass is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | insufficient recovery of nuclear DOT1L | check nuclear extraction and a nuclear protein control |
| Band higher than expected | identity or migration difference is unestablished | compare with a second DOT1L antibody or DOT1L depletion control |
| Band lower than expected | a different isoform is possible, but its mass is unknown | confirm identity by DOT1L depletion and check isoform expression |
| Multiple bands | isoforms 1 and 2 are listed, but distinct bands are unverified | use DOT1L depletion to identify specific bands |
| Weak or no signal | poor recovery of nuclear DOT1L | check nuclear enrichment and sample loading |
Experiment evidence
Western blot images showing DOT1L band
M00840 · DOT1L
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for DOT1L Western blot
🧪For positive controls for DOT1L in Western blot, you can use an HPA-confirmed positive sample once one is identified; none is supplied here.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: Adipose tissue is a candidate negative control, but the supplied HPA data do not identify a positive control.
HPA tissue expression evidence for DOT1L
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced DOT1L Western Blot Tips
Deeper troubleshooting and optimisation questions for DOT1L, answered from its protein features.
What should be checked when unexpected DOT1L bands appear?
Band shift · Compare them with the 164.9 kDa predicted mass and consider the isoform 2 sequence change at UniProt position 1537. DOT1L also has annotated phosphorylation sites, but those annotations alone cannot assign an unexpected band or prove a shift. Check antibody recognition and sample preparation before identifying a band as a DOT1L isoform or modified form.
Could DOT1L isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, the canonical asparagine at UniProt position 1537 is replaced by a longer alternative sequence. Different migration is plausible, but the supplied features give no observed band positions. Check which isoforms the antibody can recognize before assigning bands.
Which DOT1L phosphorylation sites are annotated?
PTM · UniProt lists phosphoserines at positions 297, 374, 448, 471, 775, 786, 826, 834, 902, 997, 1001, 1009, 1035, 1093, 1104, 1213 and 1246; phosphothreonines at 480, 900 and 984. These are UniProt coordinates; antibody or paper numbering may differ. The annotations do not establish a visible band shift.
Does this guide establish induction of DOT1L?
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 DOT1L Western blot?
Transfer · DOT1L’s predicted mass is 164.9 kDa. Choose and optimize a transfer setup for a protein of that size, then check transfer near 165 kDa with a size marker and membrane stain. The supplied features do not identify one transfer method or settings as optimal.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M00840 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 DOT1L bands be quantified?
Quantitation · DOT1L is annotated as nuclear and has two isoforms. Compare equivalent sample preparations, use a loading measure appropriate to those preparations, and keep the band selection consistent across lanes. If multiple bands appear, establish which ones the antibody recognizes before combining their signals.
How should a band differ from DOT1L’s predicted mass be interpreted?
Interpretation · The supplied predicted mass is 164.9 kDa, but no observed band position is available. DOT1L has two isoforms and 20 annotated phosphorylation sites. Those features alone do not establish a visible shift or explain a mass difference. Check the antibody’s target region and compare bands with an appropriate size marker.
UniProt attributes phosphorylation by MAPK11 to serines 834, 902, 1001, 1009 and 1104, and threonines 900 and 984. These are UniProt coordinates. If comparing phosphorylation conditions, keep sample handling consistent and use a site-specific readout to assess a particular site; a total DOT1L band alone cannot identify its phosphorylation state.
UniProt attributes phosphorylation by MAPK11 to serines 834, 902, 1001, 1009 and 1104, and threonines 900 and 984. These are UniProt coordinates. If comparing phosphorylation conditions, keep sample handling consistent and use a site-specific readout to assess a particular site; a total DOT1L band alone cannot identify its phosphorylation state.
Boster reagents
DOT1L 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-KMT4 / Dot1L Rabbit Monoclonal Antibody
Cat # M00840
M00840 is an anti-DOT1L rabbit monoclonal antibody listed for human and mouse reactivity. Its Western blot image shows KMT4/Dot1L expression in RAW264.7 cell lysate; the supplied evidence does not document other sample contexts.
Which to pick: M00840 is the only listed option. It has a Western blot image from RAW264.7 cell lysate and is catalogued for human and mouse reactivity; check suitability for your sample and conditions.
Source: BosterBio DOT1L gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q8TEK3.
- Human Protein Atlas. DOT1L tissue expression.
- PMC8802720 — target-verified WB comparison
- PMC12804774 — target-verified WB comparison
- PMC10516645 — target-verified WB comparison