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- Table of Contents
KDM3A / Lysine-specific demethylase 3A · Western blot design guide
Design a Western Blot for KDM3A
Real validated KDM3A Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-KDM3A 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
KDM3A 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 | ~147.3 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 + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated KDM3A Western Blot Protocols
The A03994-1 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 % | 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 | A03994-1 · 2 μg/mL (catalog A03994-1) |
| Primary incubation | 1h (catalog A03994-1) |
| 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 KDM3A Western Blot Band Size?
KDM3A is predicted at 147.3 kDa; its listed features do not demonstrate altered migration, and no empirical band size is supplied.
| Band near 147.3 kDa | Consistent with predicted KDM3A size; confirm its identity |
| Band near 147.3 kDa in a nuclear fraction | Consistent with localization in round spermatids |
| Band near 147.3 kDa in a cytoplasmic fraction | Consistent with localization in elongating spermatids |
| Nuclear band weakens as a cytoplasmic band appears | Consistent with the reported localization change during spermatid elongation |
💡Expected KDM3A appearanceKDM3A has a predicted mass of 147.3 kDa, but no empirical band size is supplied; confirm any candidate band with an ordinary identity control.
| UniProt predicted mass | Places full-length KDM3A near 147.3 kDa |
| Full-length molecular weight of 147341 Da | Corresponds to the predicted 147.3 kDa size |
| Predicted mass of the 1321-residue protein | Describes the full-length sequence without establishing its observed migration |
| Predicted KDM3A mass in either cellular fraction | Remains 147.3 kDa regardless of reported nuclear or cytoplasmic localization |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | KDM3A localization varies with spermatid stage | Check sample stage and examine nuclear and cytoplasmic fractions |
| Band higher than expected | The supplied features do not establish a higher migrating form | Verify identity with KDM3A depletion or an independent antibody |
| Band lower than expected | No supplied cleavage feature explains a smaller band | Check sample integrity and verify identity with KDM3A depletion |
| Multiple bands | The supplied record lists one isoform and does not identify additional bands | Compare bands after KDM3A depletion |
| Weak or no signal | The sampled fraction may miss its stage dependent localization | Examine both nuclear and cytoplasmic fractions and verify protein recovery |
Experiment evidence
Western blot images showing KDM3A band
A03994-1 · KDM3A
A03994-1 · KDM3A
A03994-2 · KDM3A
A03994 · KDM3A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for KDM3A Western blot
🧪For positive controls for KDM3A in Western blot, you can use no HPA-validated tissue or cell line from the supplied data.
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: The supplied HPA data provide no positive or negative tissue candidates, so sample controls need validation.
HPA tissue expression evidence for KDM3A
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 KDM3A Western Blot Tips
Deeper troubleshooting and optimisation questions for KDM3A, answered from its protein features.
Where should the KDM3A band appear relative to its predicted mass?
Band shift · The predicted mass is 147.3 kDa. No observed band position is supplied, so use this as a reference rather than an expected apparent mass. The listed modifications alone do not establish a visible shift.
Could KDM3A isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It provides no isoform-based explanation for multiple bands; verify band identity before assigning them to KDM3A.
Which KDM3A modifications matter when interpreting bands?
PTM · UniProt lists phosphoserines at positions 264, 325, 445, and 766, plus N6-acetyllysine at position 895. These are UniProt coordinates; antibody or paper numbering may differ. Their presence alone does not demonstrate a visible band shift.
Does this guide establish induction of KDM3A?
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 KDM3A Western blot?
Transfer · KDM3A has a predicted mass of 147.3 kDa. Check whether protein in this size range transfers efficiently, and adjust transfer conditions if it remains in the gel. The supplied features do not specify a particular transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A03994-1 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 KDM3A be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
How should KDM3A localization guide sample preparation?
Interpretation · KDM3A is annotated in both nucleus and cytoplasm. In spermatids, it is nuclear when round and forms cytoplasmic foci during elongation; those foci disappear in mature spermatozoa. Choose the fraction and developmental stage that match the question being tested.
Compare equivalent sample types and fractions: KDM3A can occur in both nucleus and cytoplasm, and its localization changes during spermatid development. A change in one fraction need not represent a change in total cellular abundance.
Start with the 147.3 kDa predicted mass, then verify band identity. The record lists one isoform, no alternative sequence, and no glycosylation sites. Its phosphorylation and acetylation sites do not, by themselves, explain an unexpected band or a difference from predicted mass.
Compare equivalent sample types and fractions: KDM3A can occur in both nucleus and cytoplasm, and its localization changes during spermatid development. A change in one fraction need not represent a change in total cellular abundance.
Start with the 147.3 kDa predicted mass, then verify band identity. The record lists one isoform, no alternative sequence, and no glycosylation sites. Its phosphorylation and acetylation sites do not, by themselves, explain an unexpected band or a difference from predicted mass.
Boster reagents
KDM3A 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-JMJD1A KDM3A Antibody
Cat # A03994-1
Real WB data
Anti-JMJD1A/KDM3A Antibody
Cat # A03994-2
Real WB data
Anti-JMJD1A KDM3A Monoclonal Antibody
Cat # A03994
Three the supplier anti-KDM3A antibodies have Western blot images using human cell lysates. A03994-2 shows a band near 150 kDa in K562 lysate; A03994 uses HeLa and HepG2 lysates. A03994-1 reports human cell lines without naming them. Mouse and rat WB samples are not shown.
Which to pick: For human K562 lysate, A03994-2 offers a documented band and protocol. For HeLa or HepG2 lysate, consider the monoclonal A03994. A03994-1 lists human and rat reactivity; A03994-2 lists human and mouse, but the supplied WB evidence is from human samples.
Source: BosterBio KDM3A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9Y4C1.
- Human Protein Atlas. KDM3A tissue expression.
- PMC12834307 — target-verified WB comparison
- PMC9118730 — target-verified WB comparison
- PMC8157436 — target-verified WB comparison