This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
EBI3 / Interleukin-27 subunit beta · Western blot design guide
Design a Western Blot for EBI3
Real validated EBI3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-EBI3 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
EBI3 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 | ~25.4 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Placenta (IHC candidate; verify WB) | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
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 | 1 isoform(s) |
Section 1
Real Curated EBI3 Western Blot Protocols
The A07190-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Target-positive lysate and matched negative control (standard starting point) |
| Gel % | 12–15% (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 | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A07190-1 · 2 μg/mL (catalog A07190-1) |
| Primary incubation | 1h (catalog A07190-1) |
| 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 EBI3 Western Blot Band Size?
EBI3 has a predicted 25.4 kDa precursor; signal-peptide cleavage and two N-linked glycosylation sites may affect migration, but no empirical band size is supplied.
| Band near 25.4 kDa | consistent with the predicted precursor mass; identity needs confirmation |
| Band above 25.4 kDa | may reflect N-linked glycosylation at Asn55 or Asn105; migration is unverified |
| Band below 25.4 kDa | may reflect cleavage of the 1–20 signal peptide |
| Little or no band in whole-cell lysate | consistent with EBI3 secretion |
💡Expected EBI3 appearanceUniProt predicts a 25.4 kDa precursor; signal-peptide cleavage and N-linked glycosylation may affect migration, but no empirical band size is supplied, so confirm band identity with appropriate controls.
| Predicted precursor mass | 25.4 kDa provides the sequence-based reference |
| N-linked glycosylation at Asn55 | may increase apparent size; the shift is not established |
| N-linked glycosylation at Asn105 | may increase apparent size; the shift is not established |
| Signal peptide at residues 1–20 | cleavage may yield a smaller mature protein than the precursor |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | EBI3 is secreted | check conditioned medium alongside lysate |
| Band higher than expected | N-linked glycosylation is possible | compare untreated and deglycosylated samples |
| Band lower than expected | signal-peptide cleavage may lower mass | compare with a verified EBI3 positive control |
| Broad smear instead of sharp band | heterogeneous glycosylation is possible but unverified | compare untreated and deglycosylated samples |
| Multiple bands | processing or glycosylation may contribute; one isoform is listed | check which bands track with an EBI3 positive control |
| Weak or no signal | secreted EBI3 may be scarce in the sampled lysate | test conditioned medium and a verified positive control |
Experiment evidence
Western blot images showing EBI3 band
A07190-1 · EBI3
M07190-2 · EBI3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for EBI3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for EBI3 in Western blot, you can use placenta tissue.
Positive control: Placenta (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: EBI3 is secreted, so whole-cell lysates may give weak signal; conditioned medium may be more suitable.
HPA tissue expression evidence for EBI3
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 |
|---|---|---|---|---|
| Placenta | cytotrophoblasts | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EBI3 Western Blot Tips
Deeper troubleshooting and optimisation questions for EBI3, answered from its protein features.
How should EBI3 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.
Does UniProt support multiple EBI3 isoform bands?
Isoforms · Only one isoform is supplied, with no alternative sequence. Do not assign multiple bands to splice isoforms on this evidence alone.
Which EBI3 glycosylation sites matter when assessing a band shift?
PTM · UniProt lists N-linked glycosylation at asparagines 55 and 105, using precursor sequence coordinates. Keep that numbering explicit when comparing antibody or paper annotations. The listed sites do not, by themselves, establish a visible shift.
Does this guide establish induction of EBI3?
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.
How should transfer be checked for EBI3?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A07190-1 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 secreted EBI3 be quantified across samples?
Quantitation · Compare the same sample fraction across conditions, since EBI3 is secreted. A lysate-only measurement may miss protein released into the medium; the supplied features do not define a secretion rate or a normalization standard.
Why might EBI3 migrate differently from its predicted 25.4 kDa?
Interpretation · The 25.4 kDa prediction includes the precursor, which has a signal peptide at residues 1–20. EBI3 also has N-linked glycosylation sites at UniProt positions 55 and 105. Processing or glycosylation could affect migration, but these features alone do not establish an apparent band size; none is supplied here.
EBI3 has a signal peptide at UniProt residues 1–20. Account for precursor versus processed protein when comparing a band with the 25.4 kDa predicted mass; the supplied features give no measured apparent mass.
EBI3 is annotated as secreted. Check the conditioned medium when assessing its expression, and keep medium and cell lysate results distinct when interpreting band intensity.
UniProt reports heterodimers with IL27A and IL12A, neither disulfide-linked. Consider these associations when investigating unexpected bands, but the annotation alone does not show that a particular band is a heterodimer. Verify band identity experimentally.
EBI3 has a signal peptide at UniProt residues 1–20. Account for precursor versus processed protein when comparing a band with the 25.4 kDa predicted mass; the supplied features give no measured apparent mass.
EBI3 is annotated as secreted. Check the conditioned medium when assessing its expression, and keep medium and cell lysate results distinct when interpreting band intensity.
UniProt reports heterodimers with IL27A and IL12A, neither disulfide-linked. Consider these associations when investigating unexpected bands, but the annotation alone does not show that a particular band is a heterodimer. Verify band identity experimentally.
Boster reagents
EBI3 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-Interleukin-27 subunit beta EBI3 Antibody
Cat # A07190-1
Real WB data
Anti-EBI3 Rabbit Monoclonal Antibody
Cat # M07190-2
Two the supplier anti-EBI3 antibodies have Western blot images. A07190-1 shows rat spleen lysate; M07190-2 shows human cell lines and rat and mouse tissues, with a reported band near 30 kDa. The images document the stated samples and conditions only.
Which to pick: For human or mouse samples, M07190-2 has a directly relevant image; for rat spleen, both have images. Both list Human, Mouse, and Rat reactivity. Choose the antibody with the sample and protocol closest to yours; these images do not establish performance in every specimen.
Source: BosterBio EBI3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q14213.
- Human Protein Atlas. EBI3 tissue expression.
- PMC11407894 — target-verified WB comparison
- PMC6414311 — target-verified WB comparison
- PMC6611226 — target-verified WB comparison