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- Table of Contents
NKX2-2 / Homeobox protein Nkx-2.2 · Western blot design guide
Design a Western Blot for NKX2-2
Real validated NKX2-2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NKX2-2 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
NKX2-2 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 | ~30.1 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Caudate (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 | — | |
| Caveat | Blocking-peptide control | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated NKX2-2 Western Blot Protocols
The A04740 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | rat kidney tissue lysate (catalog A04740) |
| 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 | Western blot analysis of NKX2-2 in rat kidney tissue lysate with NKX2-2 antibody at 1 μg/mL in (A) the absence and (B) the presence of blocking peptide (catalog A04740) |
| Primary antibody | A04740 · 1 μg/mL (catalog A04740) |
| 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 NKX2-2 Western Blot Band Size?
NKX2-2 is predicted at 30.1 kDa; no empirical band is supplied, and its listed features do not demonstrate altered migration.
| Single band near 30.1 kDa | Consistent with the predicted size of NKX2-2; confirm identity |
| Band enriched in nuclear extract | Consistent with the annotated nuclear location |
| Little band in cytoplasmic fraction | Consistent with the annotated nuclear location |
| Several bands at different sizes | Not explained by the single listed isoform; check specificity |
💡Expected NKX2-2 appearanceNKX2-2 has a predicted mass of 30.1 kDa, but no empirical band size is supplied; confirm a candidate band with a blocking peptide or another identity control.
| UniProt predicted mass | Provides a 30.1 kDa reference for a candidate band |
| UniProt molecular weight in daltons | The 30,133 Da estimate corresponds to approximately 30.1 kDa |
| Predicted full-length molecular weight | Describes the 273-residue protein at approximately 30.1 kDa |
| Predicted monomer mass | Places a candidate monomer band near 30.1 kDa |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | NKX2-2 is annotated as nuclear | Check a nuclear extract and verify loading |
| Band higher than expected | No listed feature establishes a larger form | Check band identity with a blocking peptide |
| Band lower than expected | No listed feature establishes a cleaved form | Check band identity with a blocking peptide |
| Multiple bands | Only one isoform is listed | Compare bands using a blocking-peptide control |
| Weak or no signal | The sampled fraction may contain little nuclear NKX2-2 | Test a nuclear-enriched sample and verify loading |
Experiment evidence
Western blot images showing NKX2-2 band
A04740 · NKX2-2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NKX2-2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NKX2-2 in Western blot, you can use caudate tissue.
Positive control: Caudate (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: NKX2-2 is nuclear, and its HPA signal in caudate is medium, so nuclear enrichment may help detection.
HPA tissue expression evidence for NKX2-2
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 |
|---|---|---|---|---|
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | Medium | Protein (IHC) | HPA → |
| Hippocampus | glial 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 NKX2-2 Western Blot Tips
Deeper troubleshooting and optimisation questions for NKX2-2, answered from its protein features.
Where should the NKX2-2 band appear relative to its predicted mass?
Band shift · The supplied sequence predicts 30.1 kDa. No observed band position is available, so use 30.1 kDa as a reference, not an expected apparent mass or proof of band identity.
Could NKX2-2 isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It provides no isoform-based explanation for multiple bands.
Do annotated modifications predict an NKX2-2 band shift?
PTM · No modified residues or glycosylation sites are listed. The supplied features therefore do not support assigning a shifted band to a specific modification.
Does this guide establish induction of NKX2-2?
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 NKX2-2 Western blot?
Transfer · NKX2-2 is predicted to be 30.1 kDa. Choose transfer conditions that retain proteins near this size, then check transfer in that region. The supplied features do not establish one preferred transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A04740 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 NKX2-2 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 is most relevant for NKX2-2 detection?
Interpretation · NKX2-2 is annotated as nuclear. A nuclear fraction is therefore a relevant sample to examine; verify fraction quality before interpreting a weak or absent band.
Compare signals using consistent sample preparation and loading, especially when comparing nuclear fractions. The nuclear annotation identifies a relevant compartment but does not establish expression levels in any sample.
Compare them with the 30.1 kDa sequence prediction and assess band specificity experimentally. The record lists one isoform and no modified residues; its reported interaction with OLIG2 does not by itself identify an additional Western blot band.
Compare signals using consistent sample preparation and loading, especially when comparing nuclear fractions. The nuclear annotation identifies a relevant compartment but does not establish expression levels in any sample.
Compare them with the 30.1 kDa sequence prediction and assess band specificity experimentally. The record lists one isoform and no modified residues; its reported interaction with OLIG2 does not by itself identify an additional Western blot band.
Boster reagents
NKX2-2 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-Homeobox protein Nkx-2.2 NKX2-2 Antibody
Cat # A04740
The catalog reports A04740 with reported human, mouse, and rat reactivity. Its Western blot image shows rat kidney tissue lysate tested at 1 μg/mL with and without blocking peptide. This example does not establish Western blot performance in every listed species.
Which to pick: A04740 is the only listed option and has a Western blot image from rat kidney tissue lysate. Match its reported reactivity to your sample species, while treating the rat kidney experiment as the specific demonstrated context.
Source: BosterBio NKX2-2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O95096.
- Human Protein Atlas. NKX2-2 tissue expression.
- PMC2889284 — target-verified WB comparison
- PMC7330694 — target-verified WB comparison
- PMC5399727 — target-verified WB comparison