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EPHA1 / Ephrin type-A receptor 1 · Western blot design guide
Design a Western Blot for EPHA1
Source-linked EPHA1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EPHA1 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
EPHA1 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 | ~108.1 kDa | |
| Observed band | ~113 kDa | |
| Gel | 5–20% (catalog PA1573) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Smooth muscle (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 | 3 isoform(s) |
Section 1
Source-Linked EPHA1 Western Blot Protocol Options
The PA1573 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human Colo320, human HepG2, human Caco-2, rat brain, rat lung, mouse brain, mouse lung (catalog PA1573) |
| Gel % | 5–20% (catalog PA1573) |
| Load | 30 ug; reducing conditions (catalog PA1573) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog PA1573) |
| Membrane | nitrocellulose membrane (catalog PA1573) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog PA1573) |
| Primary antibody | PA1573 · 0.5 μg/mL (catalog PA1573) |
| Primary incubation | overnight at 4°C (catalog PA1573) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog PA1573) |
| Secondary incubation | 1.5 hour at RT (catalog PA1573) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog PA1573) |
| Detection | ECL (catalog PA1573) |
Section 2
What Is the Expected EPHA1 Western Blot Band Size?
EPHA1 is predicted at 108.1 kDa and observed at ~113 kDa; the cause of the difference is not established.
| Band at ~113 kDa | Empirical EPHA1 band; confirm identity with antibody and sample controls |
| Band near 108.1 kDa | Near the predicted full-length precursor mass |
| Band below the precursor position | May reflect signal-peptide cleavage; the mature band size is not supplied |
| Band near twice the monomer size | Could reflect retained EPHA1 homodimers if they survive sample preparation |
| Additional bands at different positions | Could reflect isoforms 1, 2, and 3; distinct migration is not established |
| Little or no band in whole-cell lysate | Membrane-associated EPHA1 may be poorly recovered or detected |
💡Expected EPHA1 appearanceEPHA1 has a predicted 108.1 kDa full-length mass and an empirical band at ~113 kDa; the cause of the difference is unestablished, so confirm band identity with appropriate controls.
| Predicted full-length mass | 108.1 kDa predicted; the empirical EPHA1 band is ~113 kDa, with no established cause for the difference |
| N-linked glycosylation at Asn414 | May affect migration, but this site alone does not establish a visible shift or smear |
| Homodimer formation | Could produce a band near twice the monomer size if dimers persist during electrophoresis |
| Isoforms 1, 2, and 3 | May differ in size; their masses and separation on a blot are not supplied |
| Signal peptide at residues 1–25 | Cleavage can make the mature protein smaller than the full-length precursor |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated EPHA1 may be poorly recovered | Check membrane extraction and use a positive control lysate |
| Band higher than expected | Persistent homodimers or N-linked glycosylation may alter migration | Check reduction and compare with a validated EPHA1-positive sample |
| Band lower than expected | Signal-peptide cleavage or an isoform may contribute | Check antibody epitope coverage and compare with a validated positive sample |
| Multiple bands | Isoforms 1, 2, and 3 are annotated, but their band positions are unknown | Verify bands with an independent EPHA1 antibody or EPHA1 depletion |
| Weak or no signal | EPHA1 may be scarce in the sampled material or poorly extracted from membranes | Use a positive control and check membrane-protein recovery |
| Fragments below expected size | Sample proteolysis could generate smaller immunoreactive products | Check sample handling and confirm EPHA1 identity with an independent antibody |
Experiment evidence
Western blot images showing EPHA1 band
PA1573 · EPHA1
A03024Y605-1 · EPHA1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EPHA1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for EPHA1 in Western blot, you can use adrenal gland tissue, an HPA positive candidate with medium expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Smooth muscle (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: As a membrane protein with medium tissue expression, EPHA1 may be easier to detect in membrane-enriched lysate.
HPA tissue expression evidence for EPHA1
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Low | Protein (IHC) | HPA → |
| Salivary gland | glandular cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced EPHA1 Western Blot Tips
Deeper troubleshooting and optimisation questions for EPHA1, answered from its protein features.
How should EPHA1 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 EPHA1 isoforms produce different bands?
Isoforms · Yes. UniProt lists three isoforms. In UniProt coordinates, isoform 2 replaces residues 446–490 and lacks 491–976; isoform 3 replaces 446–474 and lacks 475–976. Check whether the antibody epitope is retained in each isoform before assigning a band.
Which EPHA1 phosphorylation sites matter for phospho-specific blots?
PTM · In UniProt coordinates, autocatalytic phosphotyrosines are at 599, 605, 781, and 930; phosphoserines are at 906 and 910. Match the antibody’s stated site and numbering convention to these coordinates. The listed sites lie in regions missing from isoforms 2 and 3.
UniProt describes an EPHA1 signaling complex formed upon activation by EFNA1 and lists autocatalytic phosphotyrosines at 599, 605, 781, and 930. Compare stimulated and unstimulated samples with a site-matched phospho antibody, alongside total EPHA1. Activation does not establish that every listed site will change under a given condition.
Quantify a site-specific phospho signal against total EPHA1 from the same samples, and compare like isoforms. UniProt lists isoforms 2 and 3 without the regions containing the reported phosphorylation sites; an antibody recognizing those isoforms could make total-protein normalization misleading.
UniProt describes an EPHA1 signaling complex formed upon activation by EFNA1 and lists autocatalytic phosphotyrosines at 599, 605, 781, and 930. Compare stimulated and unstimulated samples with a site-matched phospho antibody, alongside total EPHA1. Activation does not establish that every listed site will change under a given condition.
Quantify a site-specific phospho signal against total EPHA1 from the same samples, and compare like isoforms. UniProt lists isoforms 2 and 3 without the regions containing the reported phosphorylation sites; an antibody recognizing those isoforms could make total-protein normalization misleading.
Does this guide establish induction of EPHA1?
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 EPHA1 Western blot?
Transfer · EPHA1 is a single-pass membrane protein with a predicted mass of 108.1 kDa and an observed band near 113 kDa. Choose and verify a transfer setup suitable for a protein in that size range. The supplied features do not establish one specific transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the PA1573 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 EPHA1 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 EPHA1 run near 113 kDa instead of 108.1 kDa?
Interpretation · The observed band is approximately 113 kDa; the predicted mass is 108.1 kDa. UniProt lists a signal peptide at residues 1–25 and N-linked glycosylation at Asn414. These features may affect the processed protein, but their presence alone does not establish the cause of the apparent mass difference.
Check antibody epitope coverage against the three isoforms: isoforms 2 and 3 lack most of the canonical C-terminal sequence. Also consider the signal peptide at 1–25 and N-linked glycosylation at Asn414, using UniProt numbering. These features provide candidates to investigate; they do not by themselves identify an unexpected band or prove a visible shift.
Check antibody epitope coverage against the three isoforms: isoforms 2 and 3 lack most of the canonical C-terminal sequence. Also consider the signal peptide at 1–25 and N-linked glycosylation at Asn414, using UniProt numbering. These features provide candidates to investigate; they do not by themselves identify an unexpected band or prove a visible shift.
Boster reagents
EPHA1 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-Eph receptor A1/EPHA1 Antibody Picoband®
Cat # PA1573
Real WB data
Anti-EPHA1 (Phospho-Tyr605) Antibody
Cat # A03024Y605-1
Two the supplier EPHA1 antibodies have WB images. PA1573 shows a band near 113 kDa in human cell, rat tissue, and mouse tissue lysates. A03024Y605-1 is labeled phospho-Tyr605 and has a HeLa WB image; the supplied evidence does not establish phosphorylation specificity.
Which to pick: Choose PA1573 for total EPHA1, especially when matching its demonstrated human, rat, or mouse lysate contexts. Consider A03024Y605-1 for phospho-Tyr605 studies in human or mouse samples; its supplied WB image uses HeLa cells, with and without LPS treatment.
Source: BosterBio EPHA1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P21709.
- Human Protein Atlas. EPHA1 tissue expression.
- PMC4827226 — target-verified WB comparison
- PMC9581518 — target-verified WB comparison