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- Table of Contents
NOTCH4 / Neurogenic locus notch homolog protein 4 · Western blot design guide
Design a Western Blot for NOTCH4
Real validated NOTCH4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NOTCH4 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
NOTCH4 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 | ~209.6 kDa | |
| Observed band | 210 kDa | |
| Gel | 4–12% gradient (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated NOTCH4 Western Blot Protocols
The PA1831 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | A549 at 40ug, SMMC at 40ug, HELA at 40ug Predicted bind size: 210KD Observed bind size: 210KD (catalog PA1831) |
| 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 | PA1831; 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 NOTCH4 Western Blot Band Size?
NOTCH4 is predicted at 209.6 kDa and observed at 210 kDa; the cause of their small difference is not established.
| Band near 210 kDa | Empirical NOTCH4 band near its 209.6 kDa predicted mass; confirm identity with antibody controls |
| Band slightly below the precursor | Possible removal of the 1–23 signal peptide; the migration change is unquantified |
| Smaller bands | Possible N-terminal or C-terminal fragments of the described heterodimer; fragment masses are unknown |
| Band above 210 kDa | N-linked glycosylation could affect migration, but its visible effect is unestablished |
| Several bands | Isoforms 1, 2, and 3 or heterodimer fragments are possible; distinct isoform bands are unestablished |
💡Expected NOTCH4 appearanceNOTCH4 has a predicted mass of 209.6 kDa and an empirical band at 210 kDa in whole-cell lysates; confirm band identity with antibody controls because its listed features do not establish a migration difference.
| Predicted NOTCH4 mass | 209.6 kDa provides the sequence-based reference for the empirical 210 kDa band |
| N-linked glycosylation at Asn664 | Could alter apparent size; no site-specific shift is established |
| N-linked glycosylation at Asn714 | Could alter apparent size; no site-specific shift is established |
| N-linked glycosylation at Asn964 | Could alter apparent size; no site-specific shift is established |
| N-linked glycosylation at Asn1143 | Could alter apparent size; no site-specific shift is established |
| Signal peptide at residues 1–23 | Its removal could make the mature chain slightly smaller than the precursor |
| Isoforms 1, 2, and 3 | May differ in size, but their masses and migration are unspecified |
| Disulfide-linked N-terminal and C-terminal heterodimer fragments | Reduction could separate fragments into lower-mass bands; their masses are unspecified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated NOTCH4 may be poorly recovered | Check membrane extraction and enrich the membrane fraction |
| Band higher than expected | Occupancy of the four N-linked sites could affect migration | Compare with a deglycosylated sample and verify band identity |
| Band lower than expected | The antibody may detect a fragment of the described heterodimer | Check antibody epitope and compare reducing with nonreducing lanes |
| Broad smear instead of sharp band | Variable N-linked glycosylation is possible but unconfirmed | Compare deglycosylated material and a positive control |
| Multiple bands | Isoforms or separation of heterodimer fragments are possible | Compare reducing with nonreducing lanes and check antibody epitope |
| Weak or no signal | Membrane-associated target may be underrepresented in the preparation | Verify extraction and use a known positive lysate |
| Fragments below expected size | N-terminal and C-terminal heterodimer fragments may separate | Compare reducing with nonreducing lanes and check antibody epitope |
Experiment evidence
Western blot images showing NOTCH4 band
PA1831 · NOTCH4
M02582 · NOTCH4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NOTCH4 Western blot
🧪For positive controls for NOTCH4 in Western blot, you can use a sample with confirmed NOTCH4 expression; the supplied HPA evidence identifies none.
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: NOTCH4 is a membrane protein, and the supplied HPA data cannot establish suitable tissue controls.
HPA tissue expression evidence for NOTCH4
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 NOTCH4 Western Blot Tips
Deeper troubleshooting and optimisation questions for NOTCH4, answered from its protein features.
How should NOTCH4 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 NOTCH4 isoforms produce different bands?
Isoforms · UniProt lists three isoforms. Relative to the canonical sequence, isoform 2 lacks residues 1–1414 and isoform 3 lacks residues 378–2003. These are UniProt canonical coordinates; check which isoform contains the antibody epitope before assigning a band.
Which glycosylation sites matter when interpreting a NOTCH4 band?
PTM · UniProt lists N-linked sites at residues 664, 714, 964 and 1143, using canonical sequence coordinates. Compare treated and untreated samples if testing glycosylation, but do not attribute an observed mass difference to these sites without experimental evidence.
Does this guide establish induction of NOTCH4?
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 NOTCH4 Western blot?
Transfer · NOTCH4 is a roughly 210 kDa single-pass membrane protein. Check transfer of high-mass protein by inspecting the membrane and post-transfer gel, then adjust the transfer conditions if substantial protein remains in the gel. The supplied features do not establish one required transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the PA1831 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 NOTCH4 bands?
Quantitation · Specify whether the measurement targets full-length NOTCH4 or a fragment. Its described heterodimer and three isoforms mean that bands recognized by different epitopes need not represent the same species. Use the same antibody and band definition across samples.
Why is the NOTCH4 band near 210 kDa?
Interpretation · The observed 210 kDa band closely matches the 209.6 kDa predicted mass. NOTCH4 also has a signal peptide at residues 1–23 and four N-linked glycosylation sites, but these features alone do not establish a visible shift.
UniProt describes NOTCH4 as a probable heterodimer of N-terminal extracellular and C-terminal transmembrane fragments, probably linked by disulfide bonds. Check whether the antibody recognizes the extracellular or C-terminal region, and compare bands under reducing and nonreducing conditions before assigning a fragment.
First check the antibody epitope against the three isoforms and the described extracellular and transmembrane fragments. Then compare reducing conditions, since the fragments are probably disulfide-linked. The four N-linked sites are additional candidates to test experimentally; their presence alone does not identify the band.
UniProt describes NOTCH4 as a probable heterodimer of N-terminal extracellular and C-terminal transmembrane fragments, probably linked by disulfide bonds. Check whether the antibody recognizes the extracellular or C-terminal region, and compare bands under reducing and nonreducing conditions before assigning a fragment.
First check the antibody epitope against the three isoforms and the described extracellular and transmembrane fragments. Then compare reducing conditions, since the fragments are probably disulfide-linked. The four N-linked sites are additional candidates to test experimentally; their presence alone does not identify the band.
Boster reagents
NOTCH4 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-NOTCH4 Antibody Picoband®
Cat # PA1831
Real WB data
Anti-NOTCH4 Rabbit Monoclonal Antibody
Cat # M02582
Two the supplier anti-NOTCH4 antibodies have Western blot images: PA1831 with A549, SMMC and HELA whole-cell lysates showing a 210 kDa band, and M02582 with mouse lung lysate. No publication evidence is supplied; these examples do not establish broader sample validation.
Which to pick: Choose PA1831 for its documented A549, SMMC and HELA blot at 0.5 µg/mL if those samples match your work. Consider M02582 for mouse lung lysate; it lists human and mouse reactivity, but the supplied image shows mouse lung only.
Source: BosterBio NOTCH4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q99466.
- Human Protein Atlas. NOTCH4 tissue expression.
- PMC10238419 — target-verified WB comparison
- PMC9282052 — target-verified WB comparison
- PMC10536982 — target-verified WB comparison