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- Table of Contents
NOTCH3 / Neurogenic locus notch homolog protein 3 · Western blot design guide
Design a Western Blot for NOTCH3
Real validated NOTCH3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NOTCH3 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
NOTCH3 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 | ~243.6 kDa | |
| Gel | 4–12% gradient (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Bronchus (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Fragment-specific bands | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated NOTCH3 Western Blot Protocols
The A00485-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | A549, sp2/0, PC12 (catalog A00485-1) |
| Gel % | 4–12% gradient (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A00485-1; 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 NOTCH3 Western Blot Band Size?
NOTCH3 has a predicted full-length mass of 243.6 kDa; processing and glycosylation could affect bands, but their migration effects are not demonstrated here.
| Band near 243.6 kDa | consistent with the predicted full-length NOTCH3 precursor, pending identity controls |
| Band slightly below the precursor | may reflect removal of the 1–39 signal peptide |
| Bands below the precursor | may represent the N-terminal or C-terminal fragments described for NOTCH3 |
| Multiple bands at different positions | may reflect precursor and processed fragments; band identity requires confirmation |
| Band migrating above the predicted position | may reflect N-linked glycosylation, though a visible shift is not established |
| Little or no band in whole-cell lysate | membrane-associated NOTCH3 may be poorly recovered |
💡Expected NOTCH3 appearanceUniProt predicts a 243.6 kDa full-length NOTCH3 precursor; signal-peptide removal, glycosylation, and fragment formation could affect migration, but no empirical band size is supplied, so confirm identity with appropriate controls.
| Predicted full-length mass | 243.6 kDa is the sequence-based precursor reference, not a measured band |
| N-linked glycosylation at Asn1179, Asn1336, and Asn1438 | could alter apparent migration; the size and visibility of any shift are unknown |
| Signal peptide at residues 1–39 | its removal produces a smaller mature chain than the full-length precursor |
| N-terminal and C-terminal fragment heterodimer | processing can yield fragments below precursor size; their masses are unspecified and their disulfide linkage is probable |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | membrane-associated NOTCH3 may be poorly recovered | check membrane-protein recovery and a positive-control lysate |
| Band higher than expected | N-linked glycosylation could affect migration | compare with a deglycosylated sample and verify band identity |
| Band lower than expected | signal-peptide removal or formation of a NOTCH3 fragment | compare antibodies recognizing different regions of NOTCH3 |
| Multiple bands | full-length precursor and processed fragments may coexist | compare reducing conditions and antibodies to the N-terminal and C-terminal regions |
| Weak or no signal | limited recovery of membrane-associated NOTCH3 | check extraction, loading, and a positive-control lysate |
| Fragments below expected size | NOTCH3 contains N-terminal and C-terminal fragments | map the detected region with antibodies to each terminus |
Experiment evidence
Western blot images showing NOTCH3 band
A00485-1 · NOTCH3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NOTCH3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NOTCH3 in Western blot, you can use appendix tissue, which HPA scores High.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Bronchus (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: As a membrane protein, NOTCH3 may give a clearer signal in membrane-enriched lysate.
HPA tissue expression evidence for NOTCH3
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 |
|---|---|---|---|---|
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | glandular cells | High | Protein (IHC) | HPA → |
| Urinary bladder | urothelial cells | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Seminal vesicle | glandular cells | Not detected | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NOTCH3 Western Blot Tips
Deeper troubleshooting and optimisation questions for NOTCH3, answered from its protein features.
How should NOTCH3 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.
Could different NOTCH3 bands represent listed isoforms?
Isoforms · The supplied record lists one isoform and no alternative sequence. Do not assign multiple bands to documented isoforms on this evidence alone; consider the listed heterodimer fragments and which region the antibody recognizes.
Which NOTCH3 glycosylation sites matter when assessing a band?
PTM · UniProt lists N-linked glycosylation at Asn1179, Asn1336, and Asn1438, using UniProt sequence coordinates. These sites are relevant when comparing protein forms, but their presence alone does not predict a visible shift or explain a measured band size.
Does this guide establish induction of NOTCH3?
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 NOTCH3 Western blot?
Transfer · NOTCH3 is a 243.6 kDa predicted, single-pass membrane protein. Choose conditions that transfer high-mass proteins and check transfer across that size range with a total-protein stain. The supplied features do not specify a particular transfer buffer, membrane, or duration.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00485-1 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 multiple NOTCH3 bands?
Quantitation · Quantify the same defined band or band group across samples, and state which signal you measured. UniProt describes N-terminal and C-terminal fragments in a heterodimer, so a fragment band and a full-sequence-sized band need not represent the same protein form. Interpret results in light of the antibody’s recognized region.
Why might NOTCH3 migrate differently from its predicted 243.6 kDa mass?
Interpretation · 243.6 kDa is the predicted mass of the full sequence. UniProt lists a signal peptide at residues 1–39, three N-linked glycosylation sites, and a heterodimer comprising N-terminal and C-terminal fragments. These features affect which protein form an antibody detects, but they do not establish an apparent band size or prove a visible shift.
UniProt lists omega-N-methylarginine at residue 2174, using UniProt sequence numbering. This modification alone does not establish a resolvable Western-blot band shift. Match the antibody’s recognized region to the protein form before assigning a band to this modification.
Check whether the antibody recognizes the N-terminal or C-terminal region: UniProt describes a heterodimer of corresponding fragments, probably linked by disulfide bonds. The record also lists 111 disulfide bonds and a signal peptide at residues 1–39. These features suggest possibilities to investigate, but do not identify an unexpected band without further evidence.
UniProt lists omega-N-methylarginine at residue 2174, using UniProt sequence numbering. This modification alone does not establish a resolvable Western-blot band shift. Match the antibody’s recognized region to the protein form before assigning a band to this modification.
Check whether the antibody recognizes the N-terminal or C-terminal region: UniProt describes a heterodimer of corresponding fragments, probably linked by disulfide bonds. The record also lists 111 disulfide bonds and a signal peptide at residues 1–39. These features suggest possibilities to investigate, but do not identify an unexpected band without further evidence.
Boster reagents
NOTCH3 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-NOTCH3 Antibody
Cat # A00485-1
A00485-1 is an anti-NOTCH3 antibody listed for human, mouse, and rat reactivity. Its WB image shows A549, sp2/0, and PC12 whole cell lysates at 1:500 dilution. No publication evidence or independent validation is supplied.
Which to pick: A00485-1 is the only listed option. Its WB image documents testing in A549, sp2/0, and PC12 whole cell lysates at 1:500; assess whether those samples and conditions fit your experiment.
Source: BosterBio NOTCH3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9UM47.
- Human Protein Atlas. NOTCH3 tissue expression.
- PMC12485164 — target-verified WB comparison
- PMC12771755 — target-verified WB comparison
- PMC5024153 — target-verified WB comparison