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NOX1 · Western blot design guide
Design a Western Blot for NOX1
Real validated NOX1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NOX1 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
NOX1 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 | ~64.9 kDa | |
| Observed band | ~65 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Glycosylation affects apparent size | |
| Regulation | IFN-γ-suppressed | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated NOX1 Western Blot Protocols
Literature-validated Western blot parameters for NOX1 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | rat brain , Lane 2: mouse brain . 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-NOX1 antigen affinity purified polyclonal antibody (Catalog # PA1666) 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 Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for NOX1 at approximately 65 kDa. The expected band size for NOX1 is at 65 kDa |
| Gel % | 10–12% |
| Load | 30 ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 65 kDa |
Section 2
What Is the Expected NOX1 Western Blot Band Size?
NOX1 has a 64.9 kDa predicted backbone and runs at almost the same ~65 kDa observed on blots, since its two N-glycosylation sites add only minimal mass.
| Major band at approximately 65 kDa | closely matches the 64.9 kDa predicted mass of the NOX1 core polypeptide, indicating little net mass is added by modification |
| Band appears slightly diffuse or broadened around 65 kDa rather than perfectly sharp | the two N-linked glycosylation sites (Asn162, Asn236) introduce mild glycoform heterogeneity |
| Band sitting just above the 64.9 kDa unmodified backbone mass | carbohydrate added at the Asn162 and Asn236 glycosylation sites accounts for the small upward shift toward the ~65 kDa observed size |
| A second, distinct band at a different apparent molecular weight | co-expression of the NOH-1L and NOH-1LV splice isoforms, which differ in length and migrate differently |
| Only a single isoform-sized band present | one splice variant, NOH-1L or NOH-1LV, predominates in that particular tissue or cell type |
💡Expected NOX1 appearanceNOX1 typically appears as a band at approximately 65 kDa, matching its 64.9 kDa predicted core mass with only a modest upward nudge from N-glycosylation at Asn162 and Asn236.
| Predicted core mass (64.9 kDa) | sets the baseline backbone size, closely matching the empirical ~65 kDa band seen on blots |
| N-glycosylation at Asn162 | adds modest carbohydrate mass, nudging the apparent size slightly above the unmodified 64.9 kDa backbone |
| N-glycosylation at Asn236 | contributes additional glycan mass and can cause mild smearing or heterogeneity in the band |
| Splice isoforms NOH-1L and NOH-1LV | differ in length and can appear as separate bands or shift which band predominates depending on tissue |
| Non-glycosylated bacterial recombinant standard | runs at or below the 64.9 kDa predicted mass since it lacks the native N-glycans present on mammalian-expressed NOX1 |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | incomplete denaturation or aggregation of the multi-pass transmembrane NOX1 protein | use fresh reducing agent and adequate SDS/detergent, and avoid prolonged boiling which promotes hydrophobic aggregation of membrane proteins |
| Broad smear instead of sharp band | heterogeneous N-glycosylation at Asn162 and Asn236 producing multiple glycoforms | treat lysate with PNGase F to deglycosylate and resolve to a single sharp band near 65 kDa, and run a higher-percentage or gradient gel |
| Multiple bands | co-detection of the NOH-1L and NOH-1LV splice isoforms | confirm isoform identity with an isoform-specific antibody or transcript-level assay and note which isoform predominates in the sample |
| Weak or no signal | NOX1 has restricted, tissue-specific expression with low abundance in many common cell lines | include a known-positive tissue lysate as a control, increase total protein loaded, and extend chemiluminescent exposure |
| No band in lysate | NOX1 is a multi-pass integral membrane protein at the plasma and invadopodium membrane, so standard lysis may not solubilize it efficiently | use a detergent-rich, membrane-protein-compatible lysis buffer and avoid centrifugation steps that discard membrane fractions |
| Band lower than expected | use of a bacterially expressed, non-glycosylated recombinant NOX1 standard lacking native N-glycans | benchmark against native tissue lysate or mammalian-expressed protein rather than bacterial recombinant standards |
Experiment evidence
Western blot images showing NOX1 band
PA1666 · NOX1
PA1739 · NOX1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NOX1 Western blot
🧪For positive controls for NOX1 in Western blot, you can use lysate from a NOX1-transfected/overexpressing cell line (e.g., HEK293 cells transiently expressing NOX1), since Human Protein Atlas tissue/cell expression data for NOX1 are not available to identify a validated endogenous positive sample.
Positive control: NOX1-transfected HEK293 cells
Negative control: no HPA expression data; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin alongside a total-protein stain (stain-free imaging, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: As a multi-pass membrane protein localized to the cell membrane/invadopodia with no HPA tissue data to guide a positive or negative sample choice, control selection relies on an overexpression system paired with siRNA knockdown or a knockout line to confirm antibody specificity.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced NOX1 Western Blot Tips
Deeper troubleshooting and optimisation questions for NOX1, answered from its protein features.
Why might NOX1 run close to its predicted 64.9 kDa mass?
NOX1 has no signal peptide or propeptide cleavage and only 2 glycosylation sites, so little post-translational mass is added. The observed ~65 kDa band closely matches the 64.9 kDa predicted mass, confirming correct migration without major processing artifacts.
Could I see two bands for NOX1 isoforms?
NOX1 has two annotated isoforms, NOH-1L and NOH-1LV, generated by alternative splicing. If both are expressed in your sample, expect two closely spaced bands near 65 kDa rather than a single clean band.
Does phosphorylation affect NOX1 band migration?
NOX1 is annotated as a phosphoprotein with one modified residue. Phosphorylation of this type typically causes only a minor mobility shift on standard SDS-PAGE, so expect a single band near 65 kDa rather than a distinct larger species.
What blocking approach suits NOX1 detection?
NOX1 carries two glycosylation sites, so avoid glycan-reactive blockers if probing near these residues. 5% non-fat milk is generally suitable, but switch to BSA if using a phospho-specific antibody, since milk casein is itself phosphorylated and can raise background.
What transfer method to use for NOX1 Western blot?
NOX1 is a multi-pass transmembrane protein located at the cell membrane and invadopodium membrane, making it hydrophobic and transfer-resistant. Use wet/tank transfer with reduced methanol (around 10%) and extended transfer time to fully elute it onto PVDF.
Which loading control fits NOX1 quantitation?
Because NOX1 resides in the cell membrane and invadopodium membrane rather than the cytosol, prefer a membrane-associated loading control over GAPDH or actin, and load equal membrane-fraction protein amounts for accurate normalization.
Why might high-MW bands appear for NOX1?
NOX1 functions within a multimeric complex with NOXA1, NOXO1, RAC1, and CYBA. Incomplete denaturation or insufficient heating/reducing conditions can leave subunit associations intact, producing higher-molecular-weight bands above the ~65 kDa monomeric species.
Boster reagents
Best NOX1 Western Blot Antibodies
BosterBio's NOX1 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-NADPH oxidase 1 NOX1 Antibody Picoband®
Cat # PA1666
Real WB data
Anti-NADPH oxidase 1 NOX1 Antibody Picoband®
Cat # PA1739
Boster's anti-NOX1 antibodies (PA1666, PA1739) each come with documented western blot data, giving researchers image-validated, ready-to-use options for detecting NOX1 protein expression in western blot experiments.
Which to pick: Both PA1666 and PA1739 have real WB validation images on file. PA1739 is shown detecting a ~65kD band in HeLa and MCF-7 whole-cell lysates; PA1666 was validated on a 5-20% gradient gel. Pick based on which cell type and predicted band size matches your sample.
Source: BosterBio NOX1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9Y5S8.
- Human Protein Atlas. NOX1 tissue expression.