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- Table of Contents
EFNA1 / Ephrin-A1 · Western blot design guide
Design a Western Blot for EFNA1
Source-linked EFNA1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EFNA1 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
EFNA1 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 | ~23.8 kDa | |
| Gel | 15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Glycosylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked EFNA1 Western Blot Protocol Options
The M04656 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HUVEC cell treated with TNF alpha (catalog M04656) |
| Gel % | 15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.2 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M04656; 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 EFNA1 Western Blot Band Size?
EFNA1 has a predicted full-length mass of 23.8 kDa; processing, Asn26 glycosylation and homodimerization could affect bands, but migration has not been demonstrated.
| Band near 23.8 kDa | Compatible with the predicted full-length precursor; identity requires controls |
| Band below 23.8 kDa | Could reflect removal of the signal peptide and propeptide |
| Band with altered mobility | N-linked glycosylation at Asn26 could affect migration; its visible effect is unknown |
| Band near twice the monomer size | Could reflect a homodimer that survives sample preparation |
| Multiple bands | Could reflect isoforms 1 and 2 or processing; their separation is unknown |
| Weak band in whole-cell lysate | The GPI-anchored protein may require membrane enrichment or extraction |
💡Expected EFNA1 appearanceThe full-length precursor has a predicted mass of 23.8 kDa; cleavage, Asn26 glycosylation and isoforms may affect migration, but no empirical band size is supplied, so verify identity with controls.
| Predicted full-length mass | 23.8 kDa is the sequence-based precursor reference, not a measured band |
| N-linked glycosylation at Asn26 | May alter apparent size; the extent is unknown |
| Signal peptide and propeptide cleavage | Can make the mature protein smaller than the full-length precursor |
| Homodimer formation | Could produce a band near twice the monomer size if the complex survives preparation |
| Splice isoforms 1 and 2 | Could differ in size, but their masses and separation are unspecified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | GPI-anchored membrane localization or insufficient extraction | Check membrane enrichment and extraction, then verify with a positive control |
| Band higher than expected | Possible Asn26 glycosylation or a homodimer surviving preparation | Compare reducing and denaturing conditions and verify identity with a control |
| Band lower than expected | Possible signal-peptide and propeptide cleavage | Compare precursor and mature-target controls or use an independent antibody |
| Multiple bands | Possible processing or isoforms 1 and 2; their migration is unspecified | Check with an independent antibody and appropriate positive and negative controls |
| Weak or no signal | Low recovery of the GPI-anchored membrane protein | Assess extraction and loading with a positive control |
Experiment evidence
Western blot images showing EFNA1 band
M04656 · EFNA1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EFNA1 Western blot
🧪For positive controls for EFNA1 in Western blot, you can use a verified EFNA1-positive sample, but 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 such as Ponceau alongside the samples.
⚠️Feasibility: A positive control needs independent verification because the supplied HPA data lists no positive tissue or cell.
HPA tissue expression evidence for EFNA1
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 EFNA1 Western Blot Tips
Deeper troubleshooting and optimisation questions for EFNA1, answered from its protein features.
How should EFNA1 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 EFNA1 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2; isoform 2 lacks residues 131–152 in canonical UniProt numbering. Check whether the antibody recognizes that region before interpreting a missing or additional band. The sequence difference does not establish where either isoform will migrate.
Choose an epitope outside canonical residues 131–152, which are missing in isoform 2. Also consider that residues 1–18 are the signal peptide and 183–205 are the propeptide when selecting an antibody intended to recognize processed EFNA1. Confirm the vendor’s epitope numbering against UniProt numbering.
Choose an epitope outside canonical residues 131–152, which are missing in isoform 2. Also consider that residues 1–18 are the signal peptide and 183–205 are the propeptide when selecting an antibody intended to recognize processed EFNA1. Confirm the vendor’s epitope numbering against UniProt numbering.
How should EFNA1 glycosylation be assessed?
PTM · UniProt identifies one N-linked site, Asn26 in canonical numbering. Compare untreated and deglycosylated samples if testing whether glycosylation contributes to a band pattern. A predicted modification site alone does not prove occupancy or a visible shift.
Does this guide establish induction of EFNA1?
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 EFNA1 Western blot?
Transfer · EFNA1 is a GPI-anchored membrane protein with a predicted mass of 23.8 kDa. Use transfer conditions validated for proteins near 24 kDa and check transfer with a total-protein stain. Account for its membrane localization when preparing samples.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M04656 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 EFNA1 bands be quantified?
Quantitation · Compare matched sample fractions and loading controls: EFNA1 is GPI-anchored at the cell membrane, while UniProt also lists it as secreted. If multiple bands occur, define which band or bands are quantified and apply that choice consistently across samples.
Why might EFNA1 migrate differently from its predicted 23.8 kDa?
Interpretation · The 23.8-kDa prediction refers to the 205-residue sequence. EFNA1 has a signal peptide at 1–18, a propeptide at 183–205, and an N-linked glycosylation site at Asn26. Consider processing and glycosylation when interpreting migration, but these features alone do not establish a visible shift or its size.
Check isoform 2, which lacks canonical residues 131–152, and processing at the 1–18 signal peptide and 183–205 propeptide. EFNA1 also has two disulfide bonds and can form homodimers. Compare reducing and nonreducing samples if testing an association, but these features do not identify an observed band by themselves.
Check isoform 2, which lacks canonical residues 131–152, and processing at the 1–18 signal peptide and 183–205 propeptide. EFNA1 also has two disulfide bonds and can form homodimers. Compare reducing and nonreducing samples if testing an association, but these features do not identify an observed band by themselves.
Boster reagents
EFNA1 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 A1 Rabbit Monoclonal Antibody
Cat # M04656
The catalog reports a rabbit monoclonal anti-EFNA1 antibody for Western blotting, with reported human reactivity. Its WB image shows Ephrin A1 expression in TNF-alpha-treated HUVEC cells; the supplied evidence covers that tested context.
Which to pick: M04656 is the only listed antibody. It has a WB image using TNF-alpha-treated HUVEC cells and reported human reactivity, making it the listed option to consider for a similar sample.
Source: BosterBio EFNA1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P20827.
- Human Protein Atlas. EFNA1 tissue expression.
- PMC8369630 — target-verified WB comparison
- PMC13571821 — target-verified WB comparison
- PMC11855005 — target-verified WB comparison