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NKX3-1 / Homeobox protein Nkx-3.1 · Western blot design guide
Design a Western Blot for NKX3-1
Real validated NKX3-1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NKX3-1 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
NKX3-1 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 | ~26.4 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Prostate (IHC candidate; verify WB) +2 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 5 isoform(s) |
Section 1
Real Curated NKX3-1 Western Blot Protocols
The A30460 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 % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A30460; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| 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 NKX3-1 Western Blot Band Size?
NKX3-1 is predicted at 26.4 kDa; five isoforms could affect migration, but no distinct band sizes or empirical migration are demonstrated.
| Band near 26.4 kDa | Consistent with the predicted NKX3-1 mass; identity needs confirmation. |
| Several discrete bands | Isoforms 1 through 5 are annotated, but their migration is unknown. |
| Stronger band in a nuclear fraction | Consistent with NKX3-1 nuclear localization. |
| Weak band in whole-cell lysate but stronger in a nuclear fraction | Consistent with enrichment of nuclear NKX3-1. |
💡Expected NKX3-1 appearanceUniProt predicts 26.4 kDa for NKX3-1; no empirical band size or isoform masses are supplied, so confirm any candidate band with ordinary identity controls.
| UniProt predicted mass | Provides a 26.4 kDa reference, not a measured migration. |
| Splice isoform 1 | Its individual mass and migration are not supplied. |
| Splice isoform 2 | Its size relative to isoform 1 is unknown. |
| Splice isoform 3 | Its size relative to other isoforms is unknown. |
| Splice isoform 4 | Its size relative to other isoforms is unknown. |
| Splice isoform 5 | Its size relative to other isoforms is unknown. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear NKX3-1 may be poorly represented in the sampled fraction. | Check a nuclear fraction and verify sample loading. |
| Band higher than expected | An isoform is possible, but its migration is not supplied. | Check antibody specificity with an NKX3-1 depletion control. |
| Band lower than expected | An isoform or unrelated antibody signal is possible; no size is established. | Compare with an NKX3-1 depletion control. |
| Multiple bands | Five splice isoforms are annotated, though distinct bands are unproven. | Check which bands track with NKX3-1 depletion. |
| Weak or no signal | The sampled material may contain little nuclear NKX3-1. | Compare nuclear enrichment and verify loading and antibody performance. |
Experiment evidence
Western blot images showing NKX3-1 band
A30460 · NKX3-1
A02709-4 · NKX3-1
M02709 · NKX3-1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NKX3-1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NKX3-1 in Western blot, you can use prostate tissue lysate, where HPA reports high expression.
Positive control: Prostate (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: NKX3-1 is nuclear, so a nuclear-enriched lysate may improve detection.
HPA tissue expression evidence for NKX3-1
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 |
|---|---|---|---|---|
| Prostate | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Cervix | glandular cells | 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 NKX3-1 Western Blot Tips
Deeper troubleshooting and optimisation questions for NKX3-1, answered from its protein features.
What band size should I expect for NKX3-1?
Band shift · The canonical sequence is 234 residues with a predicted mass of 26.4 kDa. No observed band size is supplied, so use 26.4 kDa as a reference rather than an expected apparent position.
Could NKX3-1 isoforms produce different bands?
Isoforms · Five isoforms are listed. Relative to isoform 1, isoforms 2, 3, 4, and 5 lack residues 8–56, 13–87, 15–83, and 40–83, respectively. Check whether the antibody recognizes a region retained in the isoform you want to detect. These sequence differences do not establish where bands will appear.
Could modification explain a higher NKX3-1 band?
PTM · The supplied interaction annotation says TOPORS polyubiquitinates NKX3-1, and the protein carries an Ubl conjugation keyword. No modified residue is mapped in the supplied features. Polyubiquitination is a possible consideration for a higher band, but the annotation alone cannot identify or explain a band on your blot.
Does this guide establish induction of NKX3-1?
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 NKX3-1 Western blot?
Transfer · Plan transfer around the 26.4 kDa predicted canonical protein and verify retention and transfer with a suitable molecular weight marker. The supplied features do not identify a preferred membrane, transfer mode, or settings.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A30460 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 NKX3-1 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.
Which sample fraction should I use to measure NKX3-1?
Interpretation · NKX3-1 is annotated as nuclear. A nuclear fraction can help assess it; check fraction purity and compare samples prepared the same way. A whole-cell lysate may also contain it, but the supplied features give no expected abundance.
First compare them with the 26.4 kDa canonical prediction and the four listed sequence deletions. Also consider the TOPORS polyubiquitination annotation. Neither an alternative sequence nor a modification annotation proves the identity of a particular band; confirm antibody recognition of the relevant sequence and assess band specificity experimentally.
First compare them with the 26.4 kDa canonical prediction and the four listed sequence deletions. Also consider the TOPORS polyubiquitination annotation. Neither an alternative sequence nor a modification annotation proves the identity of a particular band; confirm antibody recognition of the relevant sequence and assess band specificity experimentally.
Boster reagents
NKX3-1 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-NKX3.1 Antibody
Cat # A30460
Real WB data
Anti-NKX3-1 Antibody Picoband®
Cat # A02709-4
Real WB data
Anti-NKX3.1 Rabbit Monoclonal Antibody
Cat # M02709
All three listed NKX3-1 antibodies have WB images. A02709-4 and M02709 show human cell lysates, with bands around 36 and 35 kDa, respectively, versus an expected 26 kDa. A30460’s caption identifies only ‘various cells,’ limiting interpretation of its image.
Which to pick: For human samples, choose by the documented context: A02709-4 shows LNCAP and Jurkat lysates; M02709 shows seven human cell lines. A30460 lists human, mouse, and rat reactivity, but its WB caption does not identify the tested cells or species.
Source: BosterBio NKX3-1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q99801.
- Human Protein Atlas. NKX3-1 tissue expression.
- PMC3740340 — target-verified WB comparison
- PMC7711313 — target-verified WB comparison
- PMC2674365 — target-verified WB comparison