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- Table of Contents
NTS / Neurotensin/neuromedin N · Western blot design guide
Design a Western Blot for NTS
Real validated NTS Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NTS 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
NTS 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 | ~19.8 kDa | |
| Gel | 15% (standard starting point) | |
| Positive control | Small intestine (IHC candidate; verify WB) +1 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Cleaved | |
| Caveat | Processing-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated NTS Western Blot Protocols
The A00726 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HeLa, HepG2 cells (catalog A00726) |
| Gel % | 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 | 0.2 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A00726; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | 1:20000 (catalog A00726) |
| 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 NTS Western Blot Band Size?
The NTS precursor is predicted at 19.8 kDa; signal-peptide removal and other processing could yield smaller products, but their migration is not established.
| Band near 19.8 kDa | Consistent with the predicted NTS precursor size; identity requires controls |
| Band below 19.8 kDa | Could reflect signal-peptide removal or further precursor processing |
| Smaller fragments | Could reflect cleavage at paired basic residues; fragment masses are unknown |
| Little or no band in whole-cell lysate | NTS is secreted and packaged in secretory vesicles |
💡Expected NTS appearanceThe predicted NTS precursor is 19.8 kDa; signal-peptide removal and further processing may yield smaller products, but no empirical band size is supplied, so confirm band identity with appropriate controls.
| Predicted NTS precursor mass | Places the unprocessed protein near 19.8 kDa |
| Signal peptide at residues 1–23 | Removal produces a smaller mature form; its apparent mass is not supplied |
| Cleavage at paired basic residues | Can produce smaller fragments of unspecified mass |
| Pyrrolidone carboxylic acid at residue 151 | Modifies this residue; a resolvable size change is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | NTS is secreted and packaged in secretory vesicles | Check conditioned medium or a secretory-vesicle-enriched fraction with appropriate controls |
| Band lower than expected | Signal-peptide removal or precursor processing may yield a smaller product | Compare with a precursor control and verify the antibody's recognized region |
| Multiple bands | Precursor and processed products may coexist | Check antibody specificity and compare fractions or processing conditions |
| Weak or no signal | Secreted NTS may be scarce in the sampled lysate | Check sample expression and assay conditioned medium with a positive control |
| Fragments below expected size | Cleavage at paired basic residues may generate smaller products | Verify fragment identity using an antibody to a defined NTS region |
Experiment evidence
Western blot images showing NTS band
A00726 · NTS
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NTS Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NTS in Western blot, you can use small intestine tissue.
Positive control: Small intestine (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: NTS is secreted, so whole-cell lysates may give a weak signal.
HPA tissue expression evidence for NTS
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 |
|---|---|---|---|---|
| Small intestine | endocrine cells | High | Protein (IHC) | HPA → |
| Stomach | 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 | endocrine 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 NTS Western Blot Tips
Deeper troubleshooting and optimisation questions for NTS, answered from its protein features.
How should NTS 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.
Are alternative NTS isoforms expected to explain multiple bands?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. Do not assign extra bands to annotated isoforms on this evidence; consider processing and verify band identity experimentally.
What modification should I consider when interpreting NTS bands?
PTM · UniProt lists pyrrolidone carboxylic acid at residue 151, using its 170-residue precursor numbering. Check whether an antibody targets a region retained in the species being measured. This modification alone does not establish a visible band shift.
Does this guide establish induction of NTS?
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 NTS?
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 A00726 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 I quantify NTS across samples?
Quantitation · Compare the same identified NTS band or processed species across samples. Because NTS is secreted and packaged in secretory vesicles, use consistent sample fractions and collection conditions; a precursor band and a processed product need not measure the same pool.
Should the NTS band match the predicted 19.8 kDa?
Interpretation · 19.8 kDa is the predicted mass of the 170-residue precursor. Its signal peptide spans residues 1–23, and the sequence contains paired basic residues associated with cleavage. Processing could produce smaller species, but these features alone do not establish which bands will appear. No empirical apparent band was supplied.
NTS has a signal peptide at residues 1–23 and is annotated for cleavage on pairs of basic residues. Consider precursor and processed products when assessing additional bands. Their presence and apparent sizes must be established experimentally.
NTS is secreted and packaged in secretory vesicles. Choose a sample fraction appropriate to the NTS species of interest, and keep fraction collection consistent across comparisons.
Check whether a band could reflect the 170-residue precursor or a processed product, given the signal peptide and annotation for cleavage on pairs of basic residues. The single listed isoform offers no annotated alternative sequence. Band size alone cannot establish identity; verify the detected species.
NTS has a signal peptide at residues 1–23 and is annotated for cleavage on pairs of basic residues. Consider precursor and processed products when assessing additional bands. Their presence and apparent sizes must be established experimentally.
NTS is secreted and packaged in secretory vesicles. Choose a sample fraction appropriate to the NTS species of interest, and keep fraction collection consistent across comparisons.
Check whether a band could reflect the 170-residue precursor or a processed product, given the signal peptide and annotation for cleavage on pairs of basic residues. The single listed isoform offers no annotated alternative sequence. Band size alone cannot establish identity; verify the detected species.
Boster reagents
NTS 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-Neurotensin NTS Antibody
Cat # A00726
The catalog reports A00726, an anti-NTS antibody with reported human, mouse, and rat reactivity. Its Western blot image shows HeLa and HepG2 cell samples; the caption reports a 1:20000 secondary antibody dilution. These examples do not establish validation across all listed species.
Which to pick: A00726 is the only listed NTS antibody and has a Western blot image from HeLa and HepG2 cells. Check that its reported human, mouse, and rat reactivity fits your sample; the supplied image documents only the named cell contexts.
Source: BosterBio NTS gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P30990.
- Human Protein Atlas. NTS tissue expression.
- PMC8314182 — target-verified WB comparison
- PMC7708045 — target-verified WB comparison