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- Table of Contents
NDUFA9 / NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, mitochondrial · Western blot design guide
Design a Western Blot for NDUFA9
Real validated NDUFA9 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NDUFA9 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
NDUFA9 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 | ~42.5 kDa | |
| Observed band | ~36 kDa | |
| Gel | 5–20% (catalog A07420-3) | |
| Positive control | Adrenal gland (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 | Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated NDUFA9 Western Blot Protocols
The A07420-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Caco-2, human MCF-7, rat RH35 (catalog A07420-3) |
| Gel % | 5–20% (catalog A07420-3) |
| Load | 30 ug; reducing conditions (catalog A07420-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A07420-3) |
| Membrane | nitrocellulose membrane (catalog A07420-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A07420-3) |
| Primary antibody | A07420-3 · 0.5 μg/mL (catalog A07420-3) |
| Primary incubation | overnight at 4°C (catalog A07420-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A07420-3) |
| Secondary incubation | 1.5 hour at RT (catalog A07420-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A07420-3) |
| Detection | ECL (catalog A07420-3) |
Section 2
What Is the Expected NDUFA9 Western Blot Band Size?
NDUFA9 is predicted at 42.5 kDa and observed near 36 kDa in reducing blots; the cause of the difference is not established.
| Band near 36 kDa | matches the empirical NDUFA9 band; confirm identity with appropriate controls |
| Band near 42.5 kDa | near the predicted sequence mass; identity requires confirmation |
| Band below 42.5 kDa | could reflect processing associated with the listed transit peptide, but the cause of the 36 kDa band is unestablished |
| Weak band in whole-cell lysate | mitochondrial matrix localization may limit the detectable amount |
💡Expected NDUFA9 appearanceNDUFA9 has a predicted mass of 42.5 kDa, while antibody QC shows a band near 36 kDa in reducing whole-cell blots; confirm band identity with appropriate controls.
| Predicted sequence mass | 42.5 kDa; the empirical band migrates near 36 kDa |
| Transit peptide annotation | processing could lower mass, but cleavage and its effect on migration are not established here |
| N6-succinyllysine at residue 175 | its effect on apparent band size is not established |
| N6-acetyllysine at residue 189 | its effect on apparent band size is not established |
| N6-acetyllysine at residue 370 | its effect on apparent band size is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | mitochondrial matrix NDUFA9 may be scarce in the loaded whole-cell sample | check loading and compare with a mitochondrial-enriched fraction |
| Band higher than expected | a band near 42.5 kDa may correspond to the predicted sequence mass, but its identity is unverified | compare with the 36 kDa QC band and use NDUFA9 depletion to test identity |
| Band lower than expected | the observed 36 kDa band runs below the 42.5 kDa prediction for an undetermined reason | compare with the reducing QC blot and confirm identity by NDUFA9 depletion |
| Multiple bands | the annotated transit peptide and modified residues do not establish which bands are NDUFA9 | use NDUFA9 depletion to identify specific bands |
| Weak or no signal | mitochondrial matrix NDUFA9 may be dilute in whole-cell lysate | verify loading and assess a mitochondrial-enriched sample |
Experiment evidence
Western blot images showing NDUFA9 band
A07420-3 · NDUFA9
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NDUFA9 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NDUFA9 in Western blot, you can use adrenal gland tissue, which HPA rates as highly expressing.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: HPA reports NDUFA9 as not detected in adipose tissue, but this may not guarantee a blank Western blot.
HPA tissue expression evidence for NDUFA9
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 | endocrine cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | High | 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 → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Lung | macrophages | Low | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Low | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced NDUFA9 Western Blot Tips
Deeper troubleshooting and optimisation questions for NDUFA9, answered from its protein features.
How should NDUFA9 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 NDUFA9 isoforms explain additional bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It provides no basis for assigning additional bands to NDUFA9 isoforms.
Which NDUFA9 modification sites matter when interpreting bands?
PTM · UniProt lists N6-succinyllysine at position 175 and N6-acetyllysine at positions 189 and 370. These are UniProt sequence coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible band shift.
The listed succinylation and acetylation sites do not, by themselves, explain the difference between the observed ~36 kDa band and the predicted 42.5 kDa mass. Avoid assigning that difference to a specific modification without further evidence.
The listed succinylation and acetylation sites do not, by themselves, explain the difference between the observed ~36 kDa band and the predicted 42.5 kDa mass. Avoid assigning that difference to a specific modification without further evidence.
Does this guide establish induction of NDUFA9?
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.
How should transfer be checked for NDUFA9?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A07420-3 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 NDUFA9 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.
Why is the observed NDUFA9 band ~36 kDa instead of 42.5 kDa?
Interpretation · The observed band is ~36 kDa, while the predicted mass is 42.5 kDa. UniProt lists a transit peptide keyword but supplies no cleavage coordinate, so these features do not establish the cause of the difference.
NDUFA9 is a mitochondrial matrix component of complex I. For quantitation, compare consistently prepared mitochondrial samples with equal protein loading, and measure the same band across samples.
Check whether the ~36 kDa band is reproducible and enriched with the mitochondrial sample. The supplied features list one isoform and three modification sites, but do not establish the identity of any additional band.
NDUFA9 is a mitochondrial matrix component of complex I. For quantitation, compare consistently prepared mitochondrial samples with equal protein loading, and measure the same band across samples.
Check whether the ~36 kDa band is reproducible and enriched with the mitochondrial sample. The supplied features list one isoform and three modification sites, but do not establish the identity of any additional band.
Boster reagents
NDUFA9 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-NDUFA9 Antibody Picoband®
Cat # A07420-3
The catalog reports one anti-NDUFA9 antibody, A07420-3, with a Western blot image. Its caption reports a band near 36 kDa, below the expected 43 kDa, so confirm band identity for your application. No publication evidence was supplied.
Which to pick: A07420-3 is the only listed option. It reports human, mouse, and rat reactivity, with a Western blot image using human Caco-2 and MCF-7, rat RH35, and mouse HEPA1-6 whole cell lysates.
Source: BosterBio NDUFA9 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q16795.
- Human Protein Atlas. NDUFA9 tissue expression.
- PMC8110749 — target-verified WB comparison
- PMC13230915 — target-verified WB comparison
- PMC6350377 — target-verified WB comparison