This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
NKX2-5 / Homeobox protein Nkx-2.5 · Western blot design guide
Design a Western Blot for NKX2-5
Real validated NKX2-5 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NKX2-5 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
NKX2-5 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 | ~34.9 kDa | |
| Observed band | ~35 kDa | |
| Gel | 5–20% (catalog A00738-2) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | — | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated NKX2-5 Western Blot Protocols
The A00738-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human U20S, human U251, human SiHa (catalog A00738-2) |
| Gel % | 5–20% (catalog A00738-2) |
| Load | 30 ug; reducing conditions (catalog A00738-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00738-2) |
| Membrane | nitrocellulose membrane (catalog A00738-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00738-2) |
| Primary antibody | A00738-2 · 0.5 μg/mL (catalog A00738-2) |
| Primary incubation | overnight at 4°C (catalog A00738-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00738-2) |
| Secondary incubation | 1.5 hour at RT (catalog A00738-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00738-2) |
| Detection | ECL (catalog A00738-2) |
Section 2
What Is the Expected NKX2-5 Western Blot Band Size?
NKX2-5 is predicted at 34.9 kDa and observed near 35 kDa; the small difference has no established cause in the supplied evidence.
| Band near 35 kDa | Matches the observed NKX2-5 band and its 34.9 kDa predicted mass; confirm identity with controls |
| Band near 70 kDa | Could reflect a homodimer if sample preparation does not fully dissociate it |
| Several bands | Could include splice isoforms 1, 2, and 3; distinct migration is unproven |
| Little signal in a cytoplasmic fraction | Consistent with NKX2-5 nuclear localization |
💡Expected NKX2-5 appearanceNKX2-5 has a predicted mass of 34.9 kDa and an observed band near 35 kDa; use a positive control or NKX2-5 depletion to confirm band identity.
| UniProt predicted mass | Predicts 34.9 kDa, consistent with the observed band near 35 kDa |
| Homeobox-mediated homodimer | Could yield a band near twice the monomer size if the complex survives sample preparation |
| Splice isoform 1 | Its individual mass and migration relative to isoforms 2 and 3 are not supplied |
| Splice isoform 2 | Its individual mass and migration relative to isoforms 1 and 3 are not supplied |
| Splice isoform 3 | Its individual mass and migration relative to isoforms 1 and 2 are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | NKX2-5 may be too scarce in the sampled lysate | Check a known positive sample and assess nuclear enrichment |
| Band higher than expected | Homeobox-mediated homodimer may persist if dissociation is incomplete | Compare thoroughly denatured samples and confirm band identity by NKX2-5 depletion |
| Band lower than expected | Identity or isoform assignment is uncertain without isoform masses | Compare with the 35 kDa band in a positive control and test NKX2-5 depletion |
| Multiple bands | Splice isoforms are documented, but their band positions are unknown | Use NKX2-5 depletion or isoform-specific controls to assign bands |
| Weak or no signal | A nuclear protein may be diluted in whole-cell lysate | Compare nuclear extract with a known positive sample |
Experiment evidence
Western blot images showing NKX2-5 band
A00738-2 · NKX2-5
PB9293 · NKX2-5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NKX2-5 Western blot
🧪For positive controls for NKX2-5 in Western blot, you can use a validated NKX2-5-positive sample; the supplied HPA evidence identifies no positive tissue or cell.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA provides no tissue candidates, so a positive sample needs independent validation.
HPA tissue expression evidence for NKX2-5
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced NKX2-5 Western Blot Tips
Deeper troubleshooting and optimisation questions for NKX2-5, answered from its protein features.
What band size should I expect for NKX2-5?
Band shift · The canonical 324-residue protein has a predicted mass of 34.9 kDa, consistent with the reported ~35 kDa band. This agreement does not establish the identity of every band near 35 kDa.
Could NKX2-5 isoforms produce additional bands?
Isoforms · UniProt lists three isoforms. Isoform 2 replaces residues 112–151 and lacks 152–324; isoform 3 changes residue 112 from E to A and lacks 113–324. These shorter sequences could yield lower-mass products, but the features alone do not establish which isoforms are expressed or visible on a blot.
Check the epitope against the isoform sequences. An epitope within canonical residues 152–324 is absent from isoforms 2 and 3. Residues 112–151 differ in isoform 2, while isoform 3 retains only an altered residue 112 before its deletion.
Check the epitope against the isoform sequences. An epitope within canonical residues 152–324 is absent from isoforms 2 and 3. Residues 112–151 differ in isoform 2, while isoform 3 retains only an altered residue 112 before its deletion.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of NKX2-5?
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 NKX2-5?
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 A00738-2 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 can nuclear localization affect NKX2-5 quantitation?
Quantitation · NKX2-5 is annotated in the nucleus. Use comparable nuclear extraction and sample loading across lanes, and quantify the same defined band in each sample. If multiple isoform bands are detected, report which band or bands the measurement includes.
How should I interpret unexpected lower NKX2-5 bands?
Interpretation · Consider the annotated shorter isoforms, then check whether the antibody epitope is present in each. A lower band alone cannot identify an isoform; compare its apparent size and signal with an isoform-aware detection strategy.
NKX2-5 forms homodimers through its homeobox. If a higher band appears, its oligomerization is relevant context, but the UniProt feature does not show that a dimer survives the blotting conditions or identify the band.
NKX2-5 forms homodimers through its homeobox. If a higher band appears, its oligomerization is relevant context, but the UniProt feature does not show that a dimer survives the blotting conditions or identify the band.
Boster reagents
NKX2-5 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-Nkx2.5/NKX2-5 Antibody Picoband®
Cat # A00738-2
Real WB data
Anti-Nkx2.5/NKX2-5 Antibody Picoband®
Cat # PB9293
Both listed the supplier anti-NKX2-5 antibodies have Western blot images reporting a 35 kDa band. The supplied evidence is limited to the product captions; no publication or independent validation evidence is provided.
Which to pick: For human cell lysates, A00738-2 has an image using U20S, U251 and SiHa samples. PB9293 has an image using mouse spleen, mouse cardiac muscle and HeLa lysates, and lists human and mouse reactivity. Match your sample to the reported contexts.
Source: BosterBio NKX2-5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P52952.
- Human Protein Atlas. NKX2-5 tissue expression.
- PMC8047315 — target-verified WB comparison
- PMC5210424 — target-verified WB comparison
- PMC5984619 — target-verified WB comparison