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- Table of Contents
KMO / Kynurenine 3-monooxygenase · Western blot design guide
Design a Western Blot for KMO
Source-linked KMO Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-KMO 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
KMO Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~55.8 kDa | |
| Observed band | ~50 kDa | |
| Gel | 5–20% (catalog A05469-3) | |
| Positive control | Kidney (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked KMO Western Blot Protocol Options
The A05469-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | rat kidney, rat liver, mouse kidney, mouse liver (catalog A05469-3) |
| Gel % | 5–20% (catalog A05469-3) |
| Load | 30 ug; reducing conditions (catalog A05469-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A05469-3) |
| Membrane | nitrocellulose membrane (catalog A05469-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A05469-3) |
| Primary antibody | A05469-3 · 0.5 μg/mL (catalog A05469-3) |
| Primary incubation | overnight at 4°C (catalog A05469-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A05469-3) |
| Secondary incubation | 1.5 hour at RT (catalog A05469-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A05469-3) |
| Detection | ECL (catalog A05469-3) |
Section 2
What Is the Expected KMO Western Blot Band Size?
KMO is predicted at 55.8 kDa and observed near 50 kDa in tissue blots; the cause of the difference is not established.
| Band at ~50 kDa | Empirical KMO band in tissue lysates; confirm identity with antibody controls |
| Band near 55.8 kDa | Near the predicted KMO mass; identity requires confirmation |
| Additional bands at different positions | Could reflect isoforms 1, 2, or 3; distinct migration is unproven |
| Shifted band near the main band | Could reflect N-linked glycosylation at Asn465; a visible shift is unproven |
| Weak band in a soluble fraction | KMO is a mitochondrial outer membrane protein |
💡Expected KMO appearanceKMO has a predicted mass of 55.8 kDa, while tissue Western blots show a band at ~50 kDa; the cause of this difference is unestablished, so confirm band identity with appropriate controls.
| Predicted KMO mass | 55.8 kDa calculated; the empirical tissue band is ~50 kDa, with no established cause for the difference |
| N-linked glycosylation at Asn465 | Could alter migration, but a visible shift is unproven |
| Isoform 1 | Its individual mass and migration are unspecified |
| Isoform 2 | Its size may differ from other isoforms; migration is unspecified |
| Isoform 3 | Its size may differ from other isoforms; migration is unspecified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated KMO may be poorly recovered during extraction | Check membrane recovery and include a positive tissue lysate |
| Band higher than expected | Asn465 glycosylation is possible, but the cause of a shift is unproven | Compare glycosidase-treated and untreated samples and verify band identity |
| Band lower than expected | The observed ~50 kDa band is below the 55.8 kDa prediction for an unknown reason | Confirm identity with an independent KMO antibody or depletion control |
| Multiple bands | Three annotated isoforms could contribute, but distinct bands are unproven | Check isoform expression and validate each band with an identity control |
| Weak or no signal | Membrane-associated KMO may be underrepresented in the prepared fraction | Assess membrane extraction and compare with a positive tissue lysate |
Experiment evidence
Western blot images showing KMO band
A05469-3 · KMO
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for KMO Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for KMO in Western blot, you can use kidney tissue lysate.
Positive control: Kidney (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: As an outer mitochondrial membrane protein, KMO may require effective membrane protein extraction for a clear signal.
HPA tissue expression evidence for KMO
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 |
|---|---|---|---|---|
| Kidney | proximal tubules (cell body) | High | Protein (IHC) | HPA → |
| Endometrium | glandular cells | Medium | Protein (IHC) | HPA → |
| Liver | hepatocytes | Medium | Protein (IHC) | HPA → |
| Placenta | trophoblastic cells | Medium | Protein (IHC) | HPA → |
| Spleen | cells in white pulp | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced KMO Western Blot Tips
Deeper troubleshooting and optimisation questions for KMO, answered from its protein features.
How should KMO 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.
Could KMO isoforms produce different bands?
Isoforms · Yes. Relative to isoform 1, isoform 2 lacks residues 367–379 and isoform 3 lacks residues 367–400, using the supplied canonical sequence numbering. These deletions could affect migration, but the features do not establish where either isoform runs on a blot.
Does KMO glycosylation explain a band shift?
PTM · UniProt annotates one N-linked glycosylation site at Asn465 in canonical sequence numbering. Its presence does not establish glycan occupancy or a visible shift. Keep that numbering convention explicit when comparing antibody or paper coordinates.
Does this guide establish induction of KMO?
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 KMO Western blot?
Transfer · For this multipass membrane protein, test a wet transfer and verify recovery around the expected band region with a total-protein stain. The supplied features do not establish one optimal transfer condition.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A05469-3 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 KMO bands be quantified?
Quantitation · Use the same sample preparation and band definition across samples. If multiple bands are present, quantify them separately until their identities are established; the three annotated isoforms do not identify individual blot bands on their own.
Why might KMO appear near 50 kDa instead of 55.8 kDa?
Interpretation · The supplied apparent band is ~50 kDa, while the predicted mass is 55.8 kDa. KMO has three isoforms and one annotated N-linked glycosylation site, but those features alone do not explain the difference. Check band identity before assigning a cause.
KMO is annotated as a multipass protein in the mitochondrial outer membrane. Check that the extraction method recovers membrane proteins, and process samples consistently before comparing band intensities.
Consider the isoform 2 deletion of residues 367–379, the isoform 3 deletion of 367–400, and the annotated N-linked site at Asn465, all in canonical numbering. None alone identifies an unexpected band. Check whether the band tracks with KMO across samples before assigning it to an isoform or modification.
KMO is annotated as a multipass protein in the mitochondrial outer membrane. Check that the extraction method recovers membrane proteins, and process samples consistently before comparing band intensities.
Consider the isoform 2 deletion of residues 367–379, the isoform 3 deletion of 367–400, and the annotated N-linked site at Asn465, all in canonical numbering. None alone identifies an unexpected band. Check whether the band tracks with KMO across samples before assigning it to an isoform or modification.
Boster reagents
KMO 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-KMO Antibody Picoband®
Cat # A05469-3
The catalog reports one anti-KMO antibody, A05469-3, for human, mouse, and rat. Its Western blot image shows rat and mouse kidney and liver lysates, with a band near 50 kDa versus the expected 56 kDa. No human sample is shown.
Which to pick: A05469-3 is the only listed choice. Its WB example uses rat and mouse kidney and liver lysates at 30 µg per lane and 0.5 µg/mL antibody. Human reactivity is listed, but the supplied image does not test a human sample.
Source: BosterBio KMO gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O15229.
- Human Protein Atlas. KMO tissue expression.
- PMC13228610 — target-verified WB comparison
- PMC4479133 — target-verified WB comparison
- PMC3969302 — target-verified WB comparison