NDUFS3 / NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial · Western blot design guide

Design a Western Blot for NDUFS3

Real validated NDUFS3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NDUFS3 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

Western blot protocol sheet for NDUFS3: expected band ~30.2 kDa, hero antibody A05867, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **NDUFS3** Western blot protocol sheet — expected band ~30.2 kDa, antibody A05867, controls and PMC citations. Open the full NDUFS3 WB guide →

Navigate to: Protocols Band Controls Antibodies

NDUFS3 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	~30.2 kDa
	Gel	12–15% (standard starting point)
	Positive control ⓘ	Appendix (IHC candidate; verify WB) +4 more
	Negative control ⓘ	Oral mucosa (IHC candidate; verify WB)

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	—
	Caveat	—
	Gene-set association	MSigDB Hallmark membership
	Isoform	2 isoform(s)

Section 1

Real Curated NDUFS3 Western Blot Protocols

The A05867 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.

Recommended Western blot protocol parameters
Sample / lysate	lysates from Hela, HepG2, mouse NIH/3T3 cell line, human brain and mouse brain tissue lysate (from left to right), (catalog A05867)
Gel %	12–15% (standard starting point)
Load	20–30 µg total protein per lane; optimize for abundance (standard starting point)
Transfer	Standard semi-dry transfer; verify efficiency (standard starting point)
Membrane	0.45 µm PVDF (standard starting point)
Blocking	5% milk or 5% BSA in TBST (standard starting point)
Primary antibody	A05867; use the WB datasheet starting dilution (standard starting point)
Primary incubation	Overnight at 4 °C (standard starting point)
Secondary antibody	goat anti-rabbit IgG, 1:5000 (catalog A05867)
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 NDUFS3 Western Blot Band Size?

UniProt predicts 30.2 kDa; listed isoforms may affect migration, but no empirical band or distinct isoform sizes are supplied.

What am I looking at on my blot?
Band near 30.2 kDa	Consistent with the UniProt predicted mass; identity still needs confirmation
Bands at different masses	Isoforms 1 and 2 are listed, but their migration is unknown
Faint band in whole-cell lysate	The mitochondrial protein may be diluted among other cellular proteins
Stronger band in a mitochondrial fraction	Consistent with its mitochondrial inner membrane location

💡Expected NDUFS3 appearanceUniProt predicts 30.2 kDa, but no empirical band size is supplied; confirm any candidate band with mitochondrial enrichment and an independent antibody or other identity control.

How each factor affects band size
UniProt predicted mass	Predicts a 30.2 kDa reference protein band
Alternative splicing	Could affect apparent size; no migration difference is supplied
Isoform 1	Its individual band size is not supplied
Isoform 2	Its individual band size is not supplied

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	Mitochondrial NDUFS3 may be scarce in the sampled lysate	Check loading and compare a mitochondrial fraction
Band higher than expected	Identity is uncertain; an isoform is possible but its size is unknown	Check with an independent antibody and isoform evidence
Band lower than expected	Identity is uncertain; an isoform is possible but its size is unknown	Check with an independent antibody and isoform evidence
Multiple bands	Isoforms 1 and 2 are listed, though distinct bands are unproven	Compare isoform evidence and verify band identity independently
Weak or no signal	NDUFS3 is located in the mitochondrial inner membrane	Check loading and compare mitochondrial enrichment

Source: UniProt O75489 Human Protein Atlas product datasheet (A05867)

Experiment evidence

Western blot images showing NDUFS3 band

A05867 · NDUFS3

A05867-2 · NDUFS3

M05867 · NDUFS3

← swipe / scroll for more images →

Real WB data behind the band-size expectations in this section.

Sample controls for NDUFS3 Western blot

🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NDUFS3 in Western blot, you can use appendix tissue, which HPA rates High.

Positive control: Appendix (IHC candidate; verify WB)

Negative control: Oral mucosa (IHC candidate; verify WB)

Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.

⚠️Feasibility: Because NDUFS3 is in the mitochondrial inner membrane, mitochondrial-enriched lysate may improve detection.

HPA tissue expression evidence for NDUFS3

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
Appendix	glandular cells	High	Protein (IHC)	HPA →
Cerebellum	cells in granular layer	High	Protein (IHC)	HPA →
Cerebral cortex	neuropil	High	Protein (IHC)	HPA →
Duodenum	glandular cells	High	Protein (IHC)	HPA →
Heart muscle	cardiomyocytes	High	Protein (IHC)	HPA →

Lower expression tissues · IHC evidence, not confirmed WB-negative controls

Tissue	Cell type	Level	Evidence	Source
Oral mucosa	squamous epithelial cells	Not detected	Protein (IHC)	HPA →
Ovary	follicle cells	Not detected	Protein (IHC)	HPA →
Prostate	glandular cells	Not detected	Protein (IHC)	HPA →
Caudate	neuronal cells	Low	Protein (IHC)	HPA →
Esophagus	squamous epithelial cells	Low	Protein (IHC)	HPA →

Section 3

Advanced NDUFS3 Western Blot Tips

Deeper troubleshooting and optimisation questions for NDUFS3, answered from its protein features.

How should NDUFS3 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 NDUFS3 isoforms produce different bands?

Isoforms · Two isoforms are listed. In isoform 2, residues 128–132 change from IVYNL to VSWEI, and residues 133–264 are missing. Isoform 2 is therefore shorter, but these features do not establish that either isoform produces a detectable band.

Is a modification-based band shift expected?

PTM · No modified residues or glycosylation sites are listed. Do not assign a shifted band to a specific modification from these features alone; they also do not rule out unlisted modifications.

Does this guide establish induction of NDUFS3?

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 NDUFS3?

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 A05867 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.

What matters when quantifying NDUFS3 bands?

Quantitation · Decide which band is being measured and apply that choice consistently. NDUFS3 has two listed isoforms, and its predicted 30.2 kDa mass does not establish the position of an observed band. The supplied features provide no condition-specific expression changes to assume during comparison.

Should NDUFS3 run at its predicted 30.2 kDa?

Interpretation · 30.2 kDa is the predicted mass; no observed Western blot band is supplied. The transit peptide keyword indicates mitochondrial targeting, but no cleavage coordinate is given, so its effect on apparent mass cannot be calculated from these features.

Compare it with the listed isoform 2 sequence, which lacks canonical residues 133–264. The transit peptide keyword is another consideration, but no cleavage site is supplied. Neither feature alone identifies an observed band; check whether the antibody recognizes a region retained in isoform 2.

NDUFS3 is listed at the mitochondrial inner membrane, on the matrix side, as a peripheral membrane protein. A mitochondrial fraction is therefore relevant when assessing its signal. Keep fraction preparation consistent across samples.

Boster reagents

NDUFS3 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 Western blot analysis of lysates from Hela, HepG2, mouse NIH/3T3 cell line, human brain and mouse brain tissue lysate (from left to right), using NDUFS3 Antibody (C-term). A05867 was diluted at 1:1000 at each lane. A goat anti-rabbit IgG H&L (HRP) at 1:5000 dilution was used as the secondary antibody. Lysates at 35ug per lane.

Western blot analysis of lysates from Hela, HepG2, mouse NIH/3T3 cell line, human brain and mouse brain tissue lysate (from left to right), using NDUFS3 Antibody (C-term). A05867 was diluted at 1:1000 at each lane. A goat anti-rabbit IgG H&L (HRP) at 1:5000 dilution was used as the secondary antibody. Lysates at 35ug per lane.

Anti-NDUFS3 Antibody (C-term)

Cat # A05867

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Real WB data

Western blot analysis of NDUFS3 using anti-NDUFS3 antibody (A05867-2). Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human 293T whole cell lysates, Lane 2: human Hela whole cell lysates, Lane 3: human MCF-7 whole cell lysates, Lane 4: human Caco-2 whole cell lysates, Lane 5: rat brain tissue lysates, Lane 6: rat kidney tissue lysates, Lane 7: mouse brain tissue lysates, Lane 8: mouse kidney tissue lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-NDUFS3 antigen affinity purified polyclonal antibody (A05867-2) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for NDUFS3 at approximately 26 kDa. The expected band size for NDUFS3 is at 30 kDa.

Anti-NDUFS3 Antibody Picoband®

Cat # A05867-2

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Real WB data

Western blot analysis of NDUFS3 using anti-NDUFS3 antibody (M05867). Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human 293T whole cell lysates, Lane 2: human MCF-7 whole cell lysates, Lane 3: human Caco-2 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-NDUFS3 antigen affinity purified monoclonal antibody (M05867) at 1:500 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for NDUFS3 at approximately 27 kDa. The expected band size for NDUFS3 is at 30 kDa.

Anti-NDUFS3 Monoclonal Antibody

Cat # M05867

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All three listed anti-NDUFS3 antibodies have WB images. A05867 shows human and mouse lysates; A05867-2 shows human, mouse and rat lysates; M05867 shows human lysates. A05867-2 and M05867 report bands around 26–27 kDa, below the expected 30 kDa.

Which to pick: Choose A05867 for the shown human and mouse cell or brain lysates, A05867-2 when rat tissue evidence matters, or M05867 for the shown human cell lysates. M05867 lists mouse and rat reactivity, but its supplied WB image shows only human samples.

Source: BosterBio NDUFS3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry O75489.
Human Protein Atlas. NDUFS3 tissue expression.
PMC6385215 — target-verified WB comparison
PMC13059122 — target-verified WB comparison
PMC8076766 — target-verified WB comparison