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- Table of Contents
EPHB4 / Ephrin type-B receptor 4 · Western blot design guide
Design a Western Blot for EPHB4
Real validated EPHB4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EPHB4 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
EPHB4 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 | ~108.3 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Peptide-blocking control | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated EPHB4 Western Blot Protocols
The A00690 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HY926 cells (catalog A00690) |
| Gel % | 8–10% (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 | A00690 · 1:2000 (catalog A00690) |
| 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 EPHB4 Western Blot Band Size?
EPHB4 is predicted at 108.3 kDa; glycosylation, signal-peptide cleavage and isoforms could affect migration, but no empirical band size or feature-specific shift is established.
| Band near 108.3 kDa | Consistent with the predicted EPHB4 mass; confirm band identity with antibody controls. |
| Band above 108.3 kDa | Could reflect N-linked glycosylation at Asn203, Asn335 or Asn426; the sites alone do not establish a visible shift. |
| Slightly lower band | Could reflect cleavage of the 1–15 signal peptide; a resolvable shift is not established. |
| Several bands at different positions | Could represent isoforms 1, 2, 3 and 4 if they migrate differently; distinct bands are not established. |
💡Expected EPHB4 appearanceEPHB4 has a predicted mass of 108.3 kDa, but no empirical band size is supplied; assess bands near that size with antibody specificity controls because the listed features do not establish a migration pattern.
| UniProt predicted mass | Places the full-length sequence at 108.3 kDa before considering processing or apparent migration. |
| N-linked glycosylation site at Asn203 | May increase apparent size if occupied; no shift is demonstrated. |
| N-linked glycosylation site at Asn335 | May increase apparent size if occupied; no shift is demonstrated. |
| N-linked glycosylation site at Asn426 | May increase apparent size if occupied; no shift is demonstrated. |
| Signal peptide at residues 1–15 | Cleavage makes the mature chain slightly smaller than the precursor; a distinct band is not established. |
| Splice isoforms 1, 2, 3 and 4 | May differ in size, but their relative masses and band positions are not supplied. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane EPHB4 may be poorly recovered during extraction. | Check membrane protein recovery and include a positive control lysate. |
| Band higher than expected | N-linked glycosylation is possible at three listed sites; its effect on migration is unverified. | Compare with a validated deglycosylated sample and confirm band identity. |
| Band lower than expected | Signal-peptide cleavage or a different isoform is possible; neither establishes a specific band position. | Check antibody epitope coverage and confirm identity with an independent antibody. |
| Multiple bands | EPHB4 has four named splice isoforms, but their migration differences are unknown. | Compare band patterns with isoform-specific controls or an independent antibody. |
| Weak or no signal | Recovery of this single-pass membrane receptor may be low. | Check extraction and loading with a membrane protein control. |
Experiment evidence
Western blot images showing EPHB4 band
A00690 · EPHB4
A00690 · EPHB4
A00690-2 · EPHB4
A00690-2 · EPHB4
A00690-3 · EPHB4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EPHB4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for EPHB4 in Western blot, you can use appendix tissue, which HPA scores High.
Positive control: Appendix (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: As a membrane protein, EPHB4 may be easier to detect in membrane-enriched lysate.
HPA tissue expression evidence for EPHB4
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 → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Liver | hepatocytes | High | Protein (IHC) | HPA → |
| Small intestine | glandular cells | High | 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 → |
| Lymph node | non-germinal center cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EPHB4 Western Blot Tips
Deeper troubleshooting and optimisation questions for EPHB4, answered from its protein features.
How should EPHB4 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.
Which EPHB4 isoforms could produce smaller bands?
Isoforms · Four isoforms are listed. Relative to the canonical sequence, isoform 3 lacks residues 307–987, isoform 4 lacks 415–987, and isoform 2 lacks 517–987; each also has a short replacement sequence before its deletion. These isoforms could differ substantially in size. Check whether the antibody epitope is retained before assigning a smaller band to an isoform.
The listed isoforms have different C-terminal extents. An antibody recognizing a region absent from isoforms 2, 3, or 4 will measure a different set of proteins from an antibody recognizing a shared region. Confirm epitope coverage and quantify comparable bands across samples; state which bands were included.
The listed isoforms have different C-terminal extents. An antibody recognizing a region absent from isoforms 2, 3, or 4 will measure a different set of proteins from an antibody recognizing a shared region. Confirm epitope coverage and quantify comparable bands across samples; state which bands were included.
Which EPHB4 phosphorylation sites can a phospho-specific blot assess?
PTM · UniProt lists phosphoserine at 769, 770, 911, and 943; phosphothreonine at 976; and phosphotyrosine at 987. These are UniProt canonical-sequence coordinates, which may differ from antibody or paper numbering. Match the antibody’s stated site to the sequence before interpreting its signal.
N-linked glycosylation is annotated at Asn203, Asn335, and Asn426 in UniProt canonical coordinates. Consider glycosylation when evaluating band mobility or heterogeneity, while avoiding a specific shift assignment without experimental evidence. Isoform 3 lacks the canonical regions containing Asn335 and Asn426; isoform 4 lacks the region containing Asn426.
N-linked glycosylation is annotated at Asn203, Asn335, and Asn426 in UniProt canonical coordinates. Consider glycosylation when evaluating band mobility or heterogeneity, while avoiding a specific shift assignment without experimental evidence. Isoform 3 lacks the canonical regions containing Asn335 and Asn426; isoform 4 lacks the region containing Asn426.
Does this guide establish induction of EPHB4?
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 EPHB4?
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 A00690 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 EPHB4 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why might EPHB4 migrate differently from its predicted 108.3 kDa mass?
Interpretation · The 108.3 kDa value is predicted from sequence. EPHB4 has a signal peptide at residues 1–15 and N-linked glycosylation sites at Asn203, Asn335, and Asn426. Processing and glycosylation may affect apparent mass, but these features alone do not establish a visible shift or explain any particular band.
Upon ligand binding, UniProt describes a heterotetramer containing an ephrin dimer and an EPHB4 receptor dimer; oligomerization is probably required for biological responses. Compare ligand-exposed and control samples when testing a response, and interpret a phospho-specific signal alongside total EPHB4. The supplied features do not establish that ligand exposure changes any particular listed phosphorylation site.
First check antibody epitope coverage against the listed isoform deletions, then consider the three annotated N-linked glycosylation sites and signal peptide at residues 1–15. These features offer possibilities, but no observed band size is supplied, and none alone identifies an unexpected band.
Upon ligand binding, UniProt describes a heterotetramer containing an ephrin dimer and an EPHB4 receptor dimer; oligomerization is probably required for biological responses. Compare ligand-exposed and control samples when testing a response, and interpret a phospho-specific signal alongside total EPHB4. The supplied features do not establish that ligand exposure changes any particular listed phosphorylation site.
First check antibody epitope coverage against the listed isoform deletions, then consider the three annotated N-linked glycosylation sites and signal peptide at residues 1–15. These features offer possibilities, but no observed band size is supplied, and none alone identifies an unexpected band.
Boster reagents
EPHB4 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-Ephrin type-B receptor 4 EphB4 Antibody
Cat # A00690
Real WB data
Anti-EphB4 (E601) Antibody
Cat # A00690-2
Real WB data
Anti-EPHB4 Antibody
Cat # A00690-3
Three the supplier anti-EPHB4 antibodies have WB images. A00690 lists human and mouse reactivity; A00690-2 and A00690-3 also list rat. The images document particular cell lysates and conditions. No publication evidence is supplied.
Which to pick: Match the listed reactivity to your sample. A00690 shows HY926 cells; A00690-2 shows MCF-7, HepG2, 3T3-L1, and PC12 lysates. A00690-3 shows a reported 108 kDa band in human HeLa and PC-3 lysates.
Source: BosterBio EPHB4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P54760.
- Human Protein Atlas. EPHB4 tissue expression.
- PMC7164363 — target-verified WB comparison
- PMC4141800 — target-verified WB comparison
- PMC4403310 — target-verified WB comparison