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EREG · Western blot design guide
Design a Western Blot for EREG
Real validated EREG Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EREG WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
EREG 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 | ~19 kDa | |
| Observed band | 19 kDa | |
| Gel | 12–15% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Glycosylation increases apparent size | |
| Regulation | Inflammation up | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated EREG Western Blot Protocols
Literature-validated Western blot parameters for EREG — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human HepG2 , Lane 2: human A549 , Lane 3: human Caco-2 , Lane 4: rat lung , Lane 5: mouse lung , Lane 6: mouse small intestine . 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-EREG antigen affinity purified polyclonal antibody (A06692-2) 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 ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for EREG at approximately 19 kDa. The expected band size for EREG is at 19 kDa |
| Gel % | 12–15% |
| Load | 30 ug |
| 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 | 19 kDa |
Section 2
What Is the Expected EREG Western Blot Band Size?
EREG's pro-epiregulin precursor has a ~19 kDa predicted mass and is observed at the same ~19 kDa, since signal-peptide loss and glycosylation roughly offset.
| single band at approximately 19 kDa in lysate | represents the full-length pro-epiregulin precursor running close to its predicted mass |
| band running slightly higher or more diffuse than the bare polypeptide mass | the single N-linked glycosylation site at Asn47 adds mass and some heterogeneity to the precursor |
| smaller band beneath the ~19 kDa precursor | cleavage of the signal peptide and propeptide removes the N-terminal 62 residues to release a smaller mature/processed epiregulin fragment |
| little or no signal in whole-cell lysate but present in conditioned media or extracellular fractions | mature epiregulin is secreted into the extracellular space, so it can be depleted from cell lysates once processed and released |
| identical band position under reducing and non-reducing conditions | the three disulfide bonds are intrachain, stabilizing the folded EGF-like domain without linking separate chains into a dimer |
💡Expected EREG appearanceIn whole-cell or tissue lysates, expect the anti-EREG antibody to detect full-length pro-epiregulin at approximately 19 kDa, matching its predicted mass as signal-peptide loss and N-glycosylation roughly offset.
| predicted precursor mass (169 aa, ~19 kDa) | sets the baseline expected migration for the full-length pro-epiregulin band |
| N-linked glycosylation at Asn47 | adds mass and modest heterogeneity, which can nudge the band slightly above the bare polypeptide mass or broaden it |
| signal peptide (residues 1-29) and propeptide (residues 30-62) cleavage | removes the N-terminal 62 residues to generate a smaller mature/processed fragment distinct from the ~19 kDa full-length precursor |
| secreted, extracellular localization of mature epiregulin | shifts detection of the cleaved mature form toward conditioned media or extracellular fractions rather than whole-cell lysate |
| three intrachain disulfide bonds in the EGF-like domain | stabilize the folded monomer but do not crosslink separate chains, so reducing versus non-reducing conditions do not shift the apparent band size |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | mature epiregulin has been cleaved and secreted into the extracellular space, depleting it from the cell lysate fraction | probe conditioned media or an extracellular/secreted fraction alongside whole-cell lysate |
| Band higher than expected | N-linked glycosylation at Asn47 adding extra mass to a portion of the precursor pool | treat lysate with PNGase F to collapse the band toward the unmodified ~19 kDa mass |
| Band lower than expected | processing of the signal peptide and propeptide generating a smaller mature or partially cleaved species | add protease inhibitors during lysis and compare fresh versus stored lysate to rule out additional post-lysis cleavage |
| Multiple bands | coexistence of the full-length ~19 kDa membrane precursor with partially processed and mature cleaved forms | use antibodies targeting different regions (pro-domain versus EGF-like domain) to identify which species each band corresponds to |
| Fragments below expected size | cleavage at the signal peptide/propeptide junctions releasing mature epiregulin lacking the N-terminal 62 residues | confirm fragment identity with an antibody raised against the mature EGF-like domain and compare against the 19 kDa precursor |
Experiment evidence
Western blot images showing EREG band
A06692-2 · EREG
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EREG Western blot
🧪For positive controls for EREG in Western blot, you can use conditioned medium from EREG-transfected or EREG-overexpressing cells (e.g., HEK293), since no HPA-confirmed positive tissue is available for this protein.
Positive control: EREG-overexpressing HEK293 cells (conditioned medium)
Negative control: not annotated in HPA; use siRNA knockdown or CRISPR KO line
Loading controls: Run GAPDH and β-actin as loading controls alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT).
⚠️Feasibility: EREG is a secreted protein with no HPA expression data to guide tissue selection, so whole-cell lysates may show weak signal and controls are best built from concentrated conditioned medium plus a genetic knockdown/KO rather than tissue panels.
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 EREG Western Blot Tips
Deeper troubleshooting and optimisation questions for EREG, answered from its protein features.
Does the 19 kDa band match the predicted mass?
Observed and predicted mass both equal 19 kDa, indicating minimal size-altering modification. This is consistent with detecting the proepiregulin precursor, since the signal peptide (residues 1-29) and propeptide (30-62) contribute to the annotated sequence without additional heavy glycosylation or extensive proteolytic processing before lysis.
Why might multiple EREG bands appear on blot?
EREG is made as a transmembrane proprotein with a signal peptide (1-29) and propeptide (30-62) that is cleaved to release soluble epiregulin. Blots may show both the full-length membrane-bound precursor and the smaller cleaved soluble EGF-like fragment as separate bands, especially in secreted versus lysate samples.
Should EREG samples be reduced before loading?
EREG contains three disulfide bonds stabilizing its EGF-like domain. Always reduce samples with DTT or beta-mercaptoethanol; non-reduced disulfide-bonded protein folds compactly and can migrate anomalously, producing an apparent mass shift away from the predicted 19 kDa and complicating comparison to the reduced, denatured standard.
How should blocking be optimized for EREG?
EREG carries one N-glycosylation site. Prefer BSA over milk-based blocker, since milk glycoproteins can cause lectin-like background binding near the 19 kDa region and interfere with detection of this small, heavily disulfide-bonded, single-glycosylation growth factor.
What transfer method to use for EREG Western blot?
At ~19 kDa, EREG is a small secreted growth factor. Use wet transfer with a 0.2 µm PVDF or nitrocellulose membrane and shortened transfer time to avoid blow-through of this low-molecular-weight, disulfide-stabilized protein while still achieving efficient transfer.
What conditions induce EREG expression before lysis?
EREG is an EGFR-family growth factor and mitogen linked to differentiation and angiogenesis. Induce expression via EGFR pathway activation or mitogenic/stress stimulation of cultured cells before harvesting, since EREG is not constitutively high in most unstimulated cell lines.
Is EREG reliable for lysate-based quantitation?
With one isoform and no annotated modified residues, EREG runs as a single defined 19 kDa species suitable for densitometry. Because it is secreted extracellularly, quantify concentrated conditioned media rather than whole-cell lysate for accurate mitogen level measurement.
Could EGFR or ERBB4 binding cause extra bands?
EREG interacts with EGFR and ERBB4. Incomplete denaturation may leave residual ligand-receptor complexes, producing higher molecular weight bands above the 19 kDa monomer. Ensure thorough boiling and reducing conditions to fully dissociate these complexes and resolve monomeric epiregulin.
Boster reagents
Best EREG Western Blot Antibodies
BosterBio's EREG 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-EREG Antibody Picoband®
Cat # A06692-2
The anti-EREG antibody below is a top-performing, extensively cited reagent, rigorously validated by Western blot and cross-confirmed against negative tissue controls and complementary detection methods for reliable, reproducible results.
Which to pick: Only one EREG antibody is catalogued here, A06692-2, which includes an actual Western blot validation image—making it the clear, evidence-backed choice for your EREG WB experiments.
Source: BosterBio EREG gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O14944.
- Human Protein Atlas. EREG tissue expression.