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ERBB3 / Receptor tyrosine-protein kinase erbB-3 · Western blot design guide
Design a Western Blot for ERBB3
Real validated ERBB3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ERBB3 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
ERBB3 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 | ~148.1 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Adrenal gland (total-target IHC; phospho state unverified) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Activation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 5 isoform(s) |
Section 1
Real Curated ERBB3 Western Blot Protocols
The P00539 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | SKBR3 cell treated with neuregulin (catalog P00539) |
| Gel % | 8–10% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% BSA in TBST (standard starting point) |
| Primary antibody | P00539; 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 ERBB3 Western Blot Band Size?
ERBB3 has a predicted 148.1 kDa precursor; glycosylation, signal peptide cleavage, isoforms and dimerization could affect migration, but no empirical band size is supplied.
| Band near 148.1 kDa | Compatible with the predicted full-length ERBB3 precursor; identity requires controls |
| Band above 148.1 kDa | Could reflect N-linked glycosylation; its migration effect is unmeasured |
| Band slightly below the precursor | Could reflect cleavage of the 1–19 signal peptide |
| Band near twice the monomer size | Could reflect a retained ERBB3 homodimer |
| Several bands at different positions | Could include isoforms 1–5, whose migration differences are unknown |
💡Expected ERBB3 appearanceUniProt predicts a 148.1 kDa ERBB3 precursor, but no empirical band size is supplied; confirm band identity with appropriate controls, especially because the lead antibody recognizes phosphorylated Y1222 ERBB3.
| Predicted precursor mass | 148.1 kDa is the sequence-based reference, not a measured band |
| N-linked glycosylation at Asn126 and other listed sites | Could increase or diversify apparent size; the migration effect is unmeasured |
| Signal peptide at residues 1–19 | Cleavage makes the mature protein smaller than the precursor |
| ERBB3 homodimer | A retained dimer could appear near twice the monomer size |
| Isoforms 1, 2, 3, 4 and 5 | May differ in size, but their masses and resolvable migration differences are unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | N-linked glycosylation or a retained homodimer | Compare deglycosylated and fully denatured samples with an ERBB3 identity control |
| Band lower than expected | Signal peptide cleavage or an alternative isoform | Check epitope coverage and compare with a total ERBB3 antibody |
| Broad smear instead of sharp band | Possible heterogeneity across multiple N-linked glycosylation sites | Compare treated and untreated samples after N-glycan removal |
| Multiple bands | Possible isoforms or differing glycosylation states | Check which bands track with ERBB3 depletion and assess glycosylation |
| Weak or no signal | The lead antibody recognizes phosphorylated Y1222 ERBB3 | Compare stimulated and unstimulated samples and probe total ERBB3 |
Experiment evidence
Western blot images showing ERBB3 band
P00539 · ERBB3
M00539 · ERBB3
PA1880 · ERBB3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ERBB3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): HPA tissue expression does not establish phosphorylation at the selected antibody epitope. Verify activation-state controls for the phospho-specific lead. HPA-IHC candidate guidance (verify in WB): For positive controls for ERBB3 in Western blot, you can use adrenal gland lysate, which has high HPA expression.
Positive control: Adrenal gland (total-target IHC; phospho state unverified)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: No supplied tissue is undetected, so use siRNA knockdown or a KO line for a clear negative control.
HPA tissue expression evidence for ERBB3
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 | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Smooth muscle | smooth muscle cells | Low | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Low | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | adipocytes | Medium | Protein (IHC) | HPA → |
Section 3
Advanced ERBB3 Western Blot Tips
Deeper troubleshooting and optimisation questions for ERBB3, answered from its protein features.
How should ERBB3 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.
Which ERBB3 isoforms could produce different bands?
Isoforms · UniProt lists five isoforms. Relative to isoform 1, isoform 2 replaces residues 141–183 and lacks 184–1342; isoform 3 changes C331 to F and lacks 332–1342; isoform 4 lacks 1–59; and isoform 5 lacks 1–643. Check whether the antibody’s epitope is retained before assigning a band to an isoform.
Which glycosylation sites matter when interpreting ERBB3 bands?
PTM · UniProt lists N-linked sites at asparagines 126, 250, 353, 408, 414, 437, 469, 522, 566 and 616. These are UniProt coordinates; antibody or paper numbering may differ. Compare the sites with the sequence retained by the isoform being tested. The annotations alone do not predict a band’s apparent mass.
UniProt annotates phosphoserine at residues 686 and 982, using its sequence coordinates. For a site-specific assay, confirm that the antibody targets the intended coordinate and that the isoform retains it. Compare the phospho signal with total ERBB3 from the same samples; phosphorylation annotation alone does not establish a visible band shift.
UniProt annotates phosphoserine at residues 686 and 982, using its sequence coordinates. For a site-specific assay, confirm that the antibody targets the intended coordinate and that the isoform retains it. Compare the phospho signal with total ERBB3 from the same samples; phosphorylation annotation alone does not establish a visible band shift.
Does this guide establish induction of ERBB3?
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 ERBB3 Western blot?
Transfer · ERBB3 isoform 1 is a single-pass cell-membrane protein with a predicted mass of 148.1 kDa. Select and check transfer conditions for a protein near that size, using a post-transfer protein stain or residual gel signal to assess transfer. The supplied features do not specify an optimal transfer method or duration.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the P00539 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 ERBB3 bands be quantified across samples?
Quantitation · Define whether the measurement is total ERBB3, one resolved isoform, or a phosphoserine-specific signal. Use an antibody whose epitope is retained in the isoforms being compared, and quantify distinct bands separately. For phosphoserine 686 or 982, compare site-specific signal with total ERBB3; isoforms 2, 3 and 5 lack both sites.
Why might ERBB3 migrate differently from its predicted 148.1 kDa mass?
Interpretation · The 148.1 kDa value is predicted. ERBB3 has a signal peptide at residues 1–19 and ten annotated N-linked glycosylation sites. These features are relevant when assessing apparent mass, but the supplied record gives no observed band size, and their presence alone does not establish a visible shift or explain a particular difference.
First compare antibody epitope coverage with the five annotated isoforms, especially the large missing regions in isoforms 2, 3 and 5. Consider the 1–19 signal peptide and the listed N-linked sites when assessing apparent size. Neither those annotations nor phosphoserine 686 and 982 prove the identity or cause of an unexpected band.
First compare antibody epitope coverage with the five annotated isoforms, especially the large missing regions in isoforms 2, 3 and 5. Consider the 1–19 signal peptide and the listed N-linked sites when assessing apparent size. Neither those annotations nor phosphoserine 686 and 982 prove the identity or cause of an unexpected band.
Boster reagents
ERBB3 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-Phospho-ErbB3 (Y1222) Rabbit Monoclonal Antibody
Cat # P00539
Real WB data
Anti-ErbB (HER3) Rabbit Monoclonal Antibody
Cat # M00539
Real WB data
Anti-ErbB 3/ERBB3 Antibody Picoband®
Cat # PA1880
All three listed antibodies have WB images: P00539 shows phospho-ERBB3 Y1222 in neuregulin-treated SKBR3 cells; M00539 shows ERBB3 in MCF-7 lysate; PA1880 shows four human cell lysates. PA1880's observed band is approximately 180 kDa, compared with an expected 148 kDa.
Which to pick: For Y1222 phosphorylation, consider P00539, which lists human reactivity. For total ERBB3, M00539 lists human and rat reactivity with an MCF-7 WB image; PA1880 lists human reactivity and provides a four-lysate WB image with detailed conditions and a band-size discrepancy.
Source: BosterBio ERBB3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P21860.
- Human Protein Atlas. ERBB3 tissue expression.
- PMC4808012 — target-verified WB comparison
- PMC5591538 — target-verified WB comparison
- PMC4408997 — target-verified WB comparison