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- Table of Contents
NTN1 / Netrin-1 · Western blot design guide
Design a Western Blot for NTN1
Source-linked NTN1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NTN1 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
NTN1 Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~67.7 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Testis (IHC candidate; verify WB) | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked NTN1 Western Blot Protocol Options
The PA1662 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | U87, COLO320 (catalog PA1662) |
| 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 | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | PA1662; 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 NTN1 Western Blot Band Size?
NTN1 has a predicted 67.7 kDa precursor; signal peptide cleavage and N-linked glycosylation may affect migration, but no empirical band size is supplied.
| Band near 67.7 kDa | consistent with the predicted precursor mass; identity needs confirmation |
| Band above 67.7 kDa | N-linked glycosylation may affect migration; the shift is not established |
| Band below 67.7 kDa | signal peptide removal may reduce mass; identity needs confirmation |
| Little or no band in whole-cell lysate | NTN1 is mainly secreted |
💡Expected NTN1 appearanceUniProt predicts a 67.7 kDa precursor, but no empirical band size is supplied; signal peptide removal and N-linked glycosylation may affect migration, so confirm band identity with appropriate controls.
| Predicted precursor mass | 67.7 kDa is the sequence-based reference, not a measured band |
| N-linked glycosylation at Asn95 | may affect apparent size; no shift is quantified |
| N-linked glycosylation at Asn116 | may affect apparent size; no shift is quantified |
| N-linked glycosylation at Asn131 | may affect apparent size; no shift is quantified |
| N-linked glycosylation at Asn417 | may affect apparent size; no shift is quantified |
| Signal peptide at residues 1–24 | cleavage yields a smaller mature protein than the precursor |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | NTN1 is mainly secreted | probe conditioned medium alongside lysate |
| Band higher than expected | N-linked glycosylation may alter migration | compare matched samples before and after N-glycan removal |
| Band lower than expected | signal peptide cleavage may reduce mass | compare with a precursor control and verify band identity |
| Broad smear instead of sharp band | glycosylation heterogeneity is possible but unconfirmed | compare matched samples before and after N-glycan removal |
| Multiple bands | precursor processing or glycosylation may contribute | compare lysate and conditioned medium and confirm specificity with NTN1 depletion |
Experiment evidence
Western blot images showing NTN1 band
PA1662 · NTN1
M04892 · NTN1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NTN1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NTN1 in Western blot, you can use testis tissue.
Positive control: Testis (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: NTN1 is mainly secreted, so conditioned medium may give a stronger signal than whole-cell lysate.
HPA tissue expression evidence for NTN1
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 |
|---|---|---|---|---|
| Testis | elongated or late spermatids | 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 NTN1 Western Blot Tips
Deeper troubleshooting and optimisation questions for NTN1, answered from its protein features.
How should NTN1 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.
Does the supplied record support multiple NTN1 isoform bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. Do not assign multiple bands to annotated isoforms from this evidence alone.
Which NTN1 glycosylation sites matter when interpreting a band?
PTM · UniProt lists N-linked sites at Asn95, Asn116, Asn131, and Asn417. These coordinates refer to the supplied full precursor sequence, including its signal peptide. If comparing antibody or paper numbering, check which sequence convention they use before assigning sites.
Does this guide establish induction of NTN1?
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 NTN1?
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 PA1662 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 NTN1 band intensity be quantified?
Quantitation · Compare bands from the same sample fraction and processing conditions. Because NTN1 is mainly secreted, lysate signal alone may not represent the amount released into medium. The supplied features provide no empirical band position for selecting a quantification target.
Why might NTN1 migrate differently from its predicted 67.7 kDa?
Interpretation · The 67.7 kDa prediction describes the precursor. NTN1 has a signal peptide at residues 1–24 and four annotated N-linked glycosylation sites. Processing and glycosylation may affect apparent mass, but these features alone do not establish a visible shift. No empirical band position is supplied.
NTN1 is mainly secreted, with cytoplasmic localization also reported. Choose the fraction that matches the intended measurement, and keep medium and lysate results separate when quantifying expression.
NTN1 has 15 annotated disulfide bonds. Comparing reducing and nonreducing samples may help assess whether disulfide-dependent structure affects migration. The feature count does not predict a particular band position or prove that an unexpected band is NTN1.
Consider the annotated signal peptide, four N-linked sites, and 15 disulfide bonds when investigating migration. None independently identifies an unexpected band. Verify band identity before attributing it to processing, glycosylation, or disulfide-dependent structure.
NTN1 is mainly secreted, with cytoplasmic localization also reported. Choose the fraction that matches the intended measurement, and keep medium and lysate results separate when quantifying expression.
NTN1 has 15 annotated disulfide bonds. Comparing reducing and nonreducing samples may help assess whether disulfide-dependent structure affects migration. The feature count does not predict a particular band position or prove that an unexpected band is NTN1.
Consider the annotated signal peptide, four N-linked sites, and 15 disulfide bonds when investigating migration. None independently identifies an unexpected band. Verify band identity before attributing it to processing, glycosylation, or disulfide-dependent structure.
Boster reagents
NTN1 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-Netrin 1/NTN1 Antibody Picoband®
Cat # PA1662
Real WB data
Anti-Netrin 1 NTN1 Monoclonal Antibody
Cat # M04892
PA1662 and M04892 are anti-NTN1 antibodies listed for human, mouse and rat. Their Western blot images show U87 and COLO320 cell lysates for PA1662, and human fetal brain lysate for M04892. These images document specific tested samples, not validation across all listed species.
Which to pick: Both listed antibodies have Western blot images. Choose PA1662 when the U87 or COLO320 lysate examples best match your experiment; choose M04892 when the human fetal brain example is more relevant. Neither image establishes performance in every listed species or sample type.
Source: BosterBio NTN1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O95631.
- Human Protein Atlas. NTN1 tissue expression.
- PMC10341717 — target-verified WB comparison
- PMC13227281 — target-verified WB comparison
- PMC12592616 — target-verified WB comparison