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EPHA4 / Ephrin type-A receptor 4 · Western blot design guide
Design a Western Blot for EPHA4
Real validated EPHA4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EPHA4 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
EPHA4 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 | ~109.9 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Cerebellum (IHC candidate; verify WB) +4 more | |
| Negative control | Adrenal gland (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated EPHA4 Western Blot Protocols
The A01860-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | lysates from Hela, MCF-7, SW480, mouse NIH/3T3 cell line, mouse brain tissue lysate (from left to right), (catalog A01860-1) |
| 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 | A01860-1; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | goat anti-rabbit IgG, 1:10000 (catalog A01860-1) |
| 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 EPHA4 Western Blot Band Size?
EPHA4 has a predicted precursor mass of 109.9 kDa; signal-peptide cleavage, N-linked glycosylation, and isoforms could affect migration, but no empirical band is supplied.
| Band near 109.9 kDa | consistent with the predicted EPHA4 precursor size; identity requires confirmation |
| Band slightly below 109.9 kDa | may reflect removal of the 1–19 signal peptide |
| Band above 109.9 kDa | may reflect N-linked glycosylation at Asn235, Asn340, Asn408, or Asn545 |
| Multiple bands at different positions | could reflect isoforms 1 and 2, though distinct migration is unestablished |
| Weak or absent band in whole-cell lysate | may reflect limited recovery of membrane-localized EPHA4 |
💡Expected EPHA4 appearanceEPHA4 has a predicted precursor mass of 109.9 kDa; signal-peptide removal and N-linked glycosylation may affect migration, but no empirical band position is supplied, so confirm identity with appropriate controls.
| Predicted precursor mass | provides a 109.9 kDa reference, not a measured band position |
| N-linked glycosylation at Asn235 | may increase apparent size; its visible effect is unestablished |
| N-linked glycosylation at Asn340 | may increase apparent size; its visible effect is unestablished |
| N-linked glycosylation at Asn408 | may increase apparent size; its visible effect is unestablished |
| N-linked glycosylation at Asn545 | may increase apparent size; its visible effect is unestablished |
| Signal peptide at residues 1–19 | cleavage makes mature EPHA4 smaller than its precursor |
| Isoforms 1 and 2 | may differ in size, but their masses and migration difference are unspecified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | membrane-localized EPHA4 may be poorly recovered | check membrane-protein extraction and an EPHA4-positive lysate |
| Band higher than expected | N-linked glycosylation may alter migration | compare with a deglycosylated aliquot and confirm band identity |
| Band lower than expected | signal-peptide removal may lower precursor mass | confirm identity with an independent EPHA4 antibody or loss-of-expression control |
| Broad smear instead of sharp band | variable N-linked glycosylation is possible but unconfirmed | compare treated and untreated aliquots after N-glycan removal |
| Multiple bands | isoforms 1 and 2 or differing glycosylation may contribute | confirm which bands track EPHA4 expression |
| Weak or no signal | membrane extraction may yield little EPHA4 | check extraction recovery and an EPHA4-positive control |
Experiment evidence
Western blot images showing EPHA4 band
A01860-1 · EPHA4
A01860-2 · EPHA4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EPHA4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for EPHA4 in Western blot, you can use cerebellum tissue, which HPA rates as highly positive.
Positive control: Cerebellum (IHC candidate; verify WB)
Negative control: Adrenal gland (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: HPA identifies a clear negative tissue, but EPHA4's membrane localization may make detection in whole-tissue lysate less reliable.
HPA tissue expression evidence for EPHA4
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 |
|---|---|---|---|---|
| Cerebellum | molecular layer cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuropil | High | Protein (IHC) | HPA → |
| Placenta | syncytiotrophoblasts - cell body | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Colon | enterocytes | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Duodenum | endocrine cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EPHA4 Western Blot Tips
Deeper troubleshooting and optimisation questions for EPHA4, answered from its protein features.
How should EPHA4 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 EPHA4 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, canonical residues 1–53 are replaced by MK. Check whether the antibody recognizes the affected N-terminal region; the sequence difference alone does not establish where either isoform will migrate.
Which EPHA4 phosphorylation sites can guide phospho-specific detection?
PTM · UniProt lists phosphotyrosines at residues 596, 602, 779, and 928, each formed by autocatalysis. These are canonical UniProt coordinates; antibody or paper numbering may differ. Match the antibody's stated site to its numbering convention.
How should ligand stimulation be assessed for EPHA4?
Induction · Upon ligand binding, EPHA4 forms a heterotetramer with an ephrin dimer and a receptor dimer; oligomerization is probably required for biological responses. Compare site-specific phosphotyrosine signal at the listed autocatalytic sites with total EPHA4 under the same conditions. The features do not establish the size or direction of a change.
What transfer method to use for EPHA4 Western blot?
Transfer · EPHA4 is a 986-residue, single-pass membrane protein with a predicted mass of 109.9 kDa. Choose transfer conditions that recover a protein in this size range and verify transfer on the membrane. UniProt features do not specify a transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A01860-1 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 can EPHA4 band intensity be quantified?
Quantitation · Measure total EPHA4 using the same antibody and exposure conditions across samples, within the assay's measurable range. For phosphosite analysis, compare the site-specific signal with total EPHA4. Keep isoform recognition in mind because isoform 2 replaces canonical residues 1–53.
Why might EPHA4 migrate differently from its predicted mass?
Interpretation · EPHA4 has a predicted mass of 109.9 kDa, a signal peptide at residues 1–19, and four N-linked glycosylation sites. These features may affect apparent mass, but they do not establish a visible shift. No observed band position is supplied.
Consider the two isoforms, the signal peptide at residues 1–19, and N-linked glycosylation at canonical residues 235, 340, 408, and 545 when checking antibody recognition. These annotations alone cannot identify an unexpected band or prove that glycosylation caused its position.
Consider the two isoforms, the signal peptide at residues 1–19, and N-linked glycosylation at canonical residues 235, 340, 408, and 545 when checking antibody recognition. These annotations alone cannot identify an unexpected band or prove that glycosylation caused its position.
Boster reagents
EPHA4 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-EPHA4 Antibody (Center)
Cat # A01860-1
Real WB data
Anti-EPHA4 Antibody Picoband®
Cat # A01860-2
Two anti-EPHA4 antibodies have Western blot images: A01860-1 with human cell lines and mouse cells and brain lysate, and A01860-2 with human cell lysates. The supplied evidence is limited to product captions; A01860-2 reports a ~135 kDa band versus 110 kDa expected.
Which to pick: For mouse samples, choose A01860-1, which lists mouse reactivity and shows mouse NIH/3T3 and brain lysate lanes. For human samples, both have Western blot images; match your sample to the reported cell lines and consider A01860-2’s band size discrepancy.
Source: BosterBio EPHA4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P54764.
- Human Protein Atlas. EPHA4 tissue expression.
- PMC6773754 — target-verified WB comparison
- PMC5541046 — target-verified WB comparison
- PMC4741646 — target-verified WB comparison