This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
EPHA5 · Western blot design guide
Design a Western Blot for EPHA5
Real validated EPHA5 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EPHA5 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
EPHA5 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 | ~114.8 kDa | |
| Observed band | ~114 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 | Heavy glycosylation load | |
| Regulation | Kras signaling dn | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated EPHA5 Western Blot Protocols
Literature-validated Western blot parameters for EPHA5 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | Rat Brain at 50ug Lane 2: Mouse Brain at 50ug. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA 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 A5 antigen affinity purified polyclonal antibody (Catalog # PB9583) 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 1:5000 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 A5 at approximately 114 kDa. The expected band size for Eph receptor A5 is at 114 kDa |
| Gel % | 8–10% |
| 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, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 114 kDa |
Section 2
What Is the Expected EPHA5 Western Blot Band Size?
EPHA5 has a 114.8 kDa predicted backbone and is empirically observed at ~114 kDa, since N-glycosylation and signal-peptide cleavage roughly offset each other.
| Single sharp band near ~114 kDa | Represents the mature, glycosylated EPHA5 receptor, whose added carbohydrate mass roughly offsets the loss from signal-peptide cleavage, landing close to the 114.8 kDa predicted mass |
| Band running slightly higher or smeared above the unmodified 114.8 kDa prediction | Reflects added mass from the six annotated N-linked glycosylation sites in the extracellular ligand-binding region |
| Band slightly smaller than an unprocessed full-length precursor | Cleavage of the 24-residue N-terminal signal peptide during membrane trafficking removes mass from the immature precursor |
| Additional fainter bands at different apparent masses | Correspond to the 3 annotated EPHA5 splice isoforms, which differ in length and domain content |
💡Expected EPHA5 appearanceExpect a single band at ~114 kDa, matching the empirically observed size and tracking the 114.8 kDa predicted mass, consistent with mature, N-glycosylated EPHA5 after signal-peptide cleavage.
| Predicted mass (114.8 kDa, 1037 aa) | sets the baseline expected molecular weight before accounting for processing or modification |
| N-glycosylation sites (six, including Asn264, Asn299, Asn369) | adds carbohydrate mass that can shift the band upward or broaden it into a smear relative to the unmodified prediction |
| Signal peptide cleavage (residues 1-24) | removes mass from the immature precursor, yielding a smaller mature-protein band than the uncleaved sequence |
| Splice isoforms 1, 2, 3 | can produce additional bands of differing apparent size depending on which isoform is expressed in the sample |
| Non-glycosylated recombinant standard | a bacterially expressed recombinant fragment lacking the native N-glycans runs lower than the native glycosylated receptor |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | EPHA5 is a single-pass transmembrane receptor that standard aqueous lysis buffers can fail to solubilize | use a detergent-based lysis buffer (e.g. RIPA) sufficient to extract membrane-anchored receptors and load a membrane-enriched fraction |
| Band higher than expected | heterogeneous occupancy of the six N-glycosylation sites adds variable carbohydrate mass | treat lysate with PNGase F to remove N-glycans and compare against untreated sample to confirm the shift |
| Broad smear instead of sharp band | heterogeneous glycoforms across the multiple N-linked glycosylation sites | run a longer gradient gel and extend transfer time, or deglycosylate the sample before loading for a sharper band |
| Multiple bands | co-expression of the 3 annotated EPHA5 splice isoforms in the tissue | confirm which isoform(s) the tissue expresses and verify the antibody epitope is present in the isoform(s) detected |
| Weak or no signal | EPHA5 expression is enriched in neural tissue (axon and dendrite projections) and may be low in non-neuronal lysates | use brain tissue lysate as a positive control and increase total protein loaded per lane |
Experiment evidence
Western blot images showing EPHA5 band
PB9583 · EPHA5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EPHA5 Western blot
🧪For positive controls for EPHA5 in Western blot, you can use brain tissue lysate or a neuronal cell line, since UniProt localizes EPHA5 to axons and dendrites, but no Human Protein Atlas expression data are available to confirm this pick.
Positive control: Brain tissue lysate
Negative control: No HPA data; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin as loading controls alongside a total-protein stain such as stain-free imaging, Ponceau S, or REVERT.
⚠️Feasibility: Because HPA has no expression data for EPHA5, there is no validated tissue to anchor positive/negative controls, so pair a plausible neuronal source with a genetic negative (siRNA knockdown or CRISPR KO) and use detergent-based lysis suited to this single-pass membrane protein.
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 EPHA5 Western Blot Tips
Deeper troubleshooting and optimisation questions for EPHA5, answered from its protein features.
Why does EPHA5 run so close to its predicted mass?
EPHA5's predicted mass is 114.8 kDa and the observed band runs at ~114 kDa, an unusually tight match. Six N-glycosylation sites add some mass, but as a single-pass type I membrane protein it can also migrate slightly anomalously on SDS-PAGE, so the two effects roughly cancel out for this receptor.
Could the three EPHA5 isoforms cause extra bands?
UniProt lists three EPHA5 isoforms from alternative splicing. Depending on which exons and antibody epitope are involved, isoforms may run at slightly different apparent masses, so a faint secondary band near the main ~114 kDa product is plausible and should not automatically be called nonspecific.
Does ephrin-A ligand binding change the EPHA5 band pattern?
EPHA5 forms a heterotetramer with dimeric ephrin-A ligand and is a receptor tyrosine kinase with four documented modified residues. Ligand stimulation drives autophosphorylation, so stimulated versus unstimulated lysates can show altered phospho-signal intensity even though the core ~114 kDa band size stays essentially unchanged under denaturing SDS-PAGE.
How should blocking be optimized for EPHA5 detection?
Because EPHA5 is a phosphoprotein with modified tyrosine residues relevant to its kinase activity, use BSA rather than milk for blocking if probing with a phospho-specific antibody, since casein in milk contains phosphoproteins that can raise background on phospho-blots.
What transfer method to use for EPHA5 Western blot?
EPHA5 is a large (114.8 kDa) single-pass transmembrane glycoprotein, so wet transfer with low or no methanol is preferred over semi-dry for efficient elution from the gel. Use PVDF membrane, given its better retention of hydrophobic transmembrane proteins, and extend transfer time to fully move this high-molecular-weight receptor.
What loading control fits EPHA5 quantitation best?
Since EPHA5 is a cell-membrane, single-pass type I membrane protein enriched in axons and dendrites, a cytosolic control like GAPDH may not track membrane-fraction loading well. Total protein normalization, or a membrane-associated control, gives a more accurate quantitation reference for this receptor.
How should unexpected higher-molecular-weight bands be interpreted?
EPHA5 has no disulfide bonds, so higher bands are not disulfide-linked dimers requiring stronger reducing conditions. Instead, bands above ~114 kDa more likely reflect incompletely denatured heterotetramer complexes formed with ephrin-A ligand, or residual aggregation typical of transmembrane proteins, rather than a distinct EPHA5 species.
Boster reagents
Best EPHA5 Western Blot Antibodies
BosterBio's EPHA5 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 A5/EPHA5 Antibody Picoband®
Cat # PB9583
These recommended anti-EPHA5 antibodies are our best-performing, most-cited Western blot reagents, thoroughly validated and orthogonally cross-validated against negative-tissue controls and complementary methods to ensure specific, reliable detection of Eph receptor A5.
Which to pick: Only one EPHA5 antibody is catalogued, PB9583; choose it by default, since it includes an actual Western blot validation image showing the Eph receptor A5 band under standard SDS-PAGE conditions.
Source: BosterBio EPHA5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P54756.
- Human Protein Atlas. EPHA5 tissue expression.