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EPHA2 · Western blot design guide
Design a Western Blot for EPHA2
Real validated EPHA2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EPHA2 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
EPHA2 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% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | N-glycosylation | |
| Regulation | p53 up | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated EPHA2 Western Blot Protocols
Literature-validated Western blot parameters for EPHA2 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human Hela , Lane 2: human U-87MG , Lane 3: human SHG-44 , Lane 4: human COLO-320 , Lane 5: human SK-OV-3 , Lane 6: human A549 , Lane 7: mouse HEPA1-6 . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Eph receptor A2 antigen affinity purified polyclonal antibody (Catalog # A00578) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for Eph receptor A2 at approximately 125KD. The expected band size for Eph receptor A2 is at 108KD |
| Gel % | 8–10% |
| Load | 50ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 125 kDa |
Section 2
What Is the Expected EPHA2 Western Blot Band Size?
EPHA2 has a 108.3 kDa calculated backbone but typically runs modestly higher due to N-glycosylation at Asn407/Asn435 and possible residual homodimer species.
| Major band running above the ~108 kDa predicted mass, sometimes appearing slightly diffuse rather than crisp | N-linked glycosylation at Asn407 and Asn435 adds carbohydrate mass and glycoform heterogeneity to the core polypeptide |
| Faint higher-molecular-weight species near roughly twice the main band, most visible without full reduction/denaturation | EPHA2 functions as a homodimer at the cell surface, and residual dimeric complex can persist if samples are incompletely reduced |
| Mature receptor band running slightly below the full calculated 108.3 kDa precursor mass | Cleavage of the N-terminal 23-residue signal peptide during maturation removes a small amount of mass from the translated precursor |
| Two (or more) distinct bands separated by a modest molecular weight difference | EPHA2 is alternatively spliced into two annotated isoforms that differ in sequence length |
| Subtle upward shift or closely spaced doublet comparing stimulated versus resting lysates | Extensive serine/threonine/tyrosine phosphorylation, including autophosphorylation upon activation, can slightly alter apparent mobility |
💡Expected EPHA2 appearanceExpect a single predominant band modestly above the 108.3 kDa calculated mass, reflecting the mature, N-glycosylated (Asn407/Asn435) receptor after signal-peptide cleavage, typically resolving as one main species under standard reducing conditions.
| Predicted mass (108.3 kDa, 976 aa) | sets the baseline backbone size before any modification is accounted for |
| N-glycosylation at Asn407 and Asn435 | adds carbohydrate mass and can broaden or shift the band above the calculated 108.3 kDa mass |
| Homodimer formation | EPHA2 dimerizes at the cell surface, so a higher-order species near twice the monomer mass may appear alongside the resolved monomer if reduction/denaturation is incomplete |
| Signal peptide cleavage (residues 1-23) | removes the N-terminal signal sequence during maturation, making the mature membrane-bound receptor slightly smaller than the full translated precursor |
| Alternative splicing (isoforms 1 and 2) | produces two receptor variants that can appear as separate bands of differing apparent size, though their individual masses are not defined here |
| Extensive Ser/Thr/Tyr phosphorylation (15 modified residues, including autophosphorylation) | can cause a subtle mobility shift or doublet appearance between resting and activated receptor pools |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | EPHA2 is a single-pass transmembrane cell-surface receptor that requires effective membrane solubilization; poor extraction leaves it in the insoluble fraction | use a detergent-based lysis buffer with sufficient incubation to solubilize membrane proteins and avoid discarding the pellet before loading |
| Band higher than expected | heterogeneous N-glycosylation at Asn407/Asn435 and/or persistence of homodimeric species under incomplete reduction | ensure samples are fully reduced and denatured with fresh reducing agent and adequate heating, and consider PNGase F treatment to collapse glycoform smearing |
| Broad smear instead of sharp band | heterogeneous glycan occupancy at the two N-glycosylation sites produces a range of apparent masses rather than one discrete band | treat lysate with PNGase F before SDS-PAGE to remove N-glycans and use a gradient gel for better resolution |
| Multiple bands | alternative splicing generates two annotated EPHA2 isoforms, and differential phosphorylation states can add closely spaced species | confirm which isoform region the antibody targets and compare against phosphatase-treated controls to distinguish splice variants from phospho-shifted forms |
| Band lower than expected | loss of the cleaved N-terminal signal peptide or reduced glycosylation in non-native expression systems yields a smaller, less modified species than native tissue receptor | compare against mammalian lysate controls and include protease inhibitors during lysis to prevent degradation |
| Weak or no signal | low native EPHA2 expression in some cell or tissue types, or loss of a phospho-specific epitope when the receptor is not actively signaling | confirm expression with a pan-EPHA2 antibody, load more total protein, and stimulate with ephrin-A1 ligand before lysis if using a phospho-specific antibody |
Experiment evidence
Western blot images showing EPHA2 band
A00578 · EPHA2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EPHA2 Western blot
🧪For positive controls for EPHA2 in Western blot, you can use a well-characterized EPHA2-expressing epithelial cell line such as A431 cells, since Human Protein Atlas tissue-level expression data are not available for this target.
Positive control: A431 cells
Negative control: no HPA-confirmed negative tissue available; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin antibodies alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: EPHA2 is a single-pass type I plasma-membrane protein rather than a secreted protein, so it should be readily detectable in standard whole-cell lysates, but with no HPA expression data to identify a validated negative tissue, specificity is best confirmed using siRNA knockdown or a CRISPR knockout line.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced EPHA2 Western Blot Tips
Deeper troubleshooting and optimisation questions for EPHA2, answered from its protein features.
Why does EPHA2 run higher than its predicted mass?
EPHA2's mature predicted mass is 108.3 kDa after signal-peptide cleavage at residue 23, but the protein carries 2 N-glycosylation sites, so observed bands commonly run higher, around 120-135 kDa. This shift reflects normal glycosylation, not degradation. If PNGase F deglycosylation is applied, the band should shift back toward the predicted 108.3 kDa mass.
Will both EPHA2 isoforms appear on the blot?
UniProt lists 2 EPHA2 isoforms (1 and 2) generated by alternative splicing. Isoform 2 is shorter and may run as a faint lower-MW band alongside the canonical ~108 kDa isoform 1, depending on whether the antibody's immunogen region overlaps both isoforms. Confirm epitope coverage before expecting dual-isoform detection.
Does ephrin-A1 stimulation increase EPHA2 phosphorylation signal?
EPHA2 has 15 modified residues, including tyrosines phosphorylated upon EFNA1 ligand binding, driving receptor autophosphorylation and recruitment of VAV2, VAV3 and PI3-kinase p85. Stimulating cells with ephrin-A1 before lysis is a practical positive control that should increase phospho-EPHA2 signal when using phospho-specific antibodies.
Should milk be avoided when blocking for phospho-EPHA2?
Yes. Because EPHA2 is heavily phosphorylated (15 modified residues) and phospho-specific detection is common, milk-based blocking is discouraged, as casein phosphoproteins can cross-react with phospho-antibodies and raise background. Use a BSA-based blocking buffer instead for cleaner phospho-EPHA2 signal and lower nonspecific binding.
What transfer method to use for EPHA2 Western blot?
EPHA2 is a large (~108-130 kDa), single-pass type I transmembrane glycoprotein, so efficient transfer favors wet/tank transfer with a low-methanol buffer and extended transfer time (60-90 min) over rapid semi-dry protocols. This ensures the high-molecular-weight membrane protein fully migrates out of the gel onto the membrane.
How should EPHA2 be normalized for quantitation?
EPHA2 localizes to the cell membrane and cell-projection ruffle membrane, so a cytosolic housekeeping protein can misrepresent membrane-fraction loading. Prefer a total-protein stain or a membrane-associated loading control instead, giving more reliable normalization across samples given EPHA2's single-pass transmembrane topology.
What causes extra EPHA2 bands under non-reducing conditions?
EPHA2 has 2 disulfide bonds and functions as a homodimer. Under non-reducing conditions, disulfide-linked dimers or aggregates can appear as higher-MW bands above the monomer. Always run reducing SDS-PAGE with DTT or beta-mercaptoethanol to resolve the expected ~108-130 kDa monomeric species and avoid misreading dimers as nonspecific bands.
Boster reagents
Best EPHA2 Western Blot Antibodies
BosterBio's EPHA2 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-Eph receptor A2/EPHA2 Antibody Picoband®
Cat # A00578
Our recommended anti-EPHA2 western blot antibodies are top-performing, extensively cited reagents validated through rigorous internal and third-party testing, including orthogonal confirmation against negative-control tissues and complementary detection methods, ensuring specific, reproducible detection of Eph receptor A2 across experimental systems.
Which to pick: Only one EPHA2 WB antibody is catalogued here: A00578, which includes an authentic western blot validation image showing specific Eph receptor A2 detection, making it the clear, single recommended choice for your experiment.
Source: BosterBio EPHA2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P29317.
- Human Protein Atlas. EPHA2 tissue expression.