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TSPAN8 / Tetraspanin-8 · Western blot design guide
Design a Western Blot for TSPAN8
Source-linked TSPAN8 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TSPAN8 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
TSPAN8 Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~26 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated | |
| Caveat | Peptide-blocking control | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked TSPAN8 Western Blot Protocol Options
The A06997 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | various cells (catalog A06997) |
| 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 | A06997 · 1:1000 (catalog A06997) |
| 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 TSPAN8 Western Blot Band Size?
TSPAN8 has a predicted mass of 26 kDa; Asn118 glycosylation and homooligomerization could affect migration, but no empirical band or feature-driven shift is demonstrated.
| Band near 26 kDa | Consistent with the predicted TSPAN8 mass; confirm identity with antibody controls. |
| Band above 26 kDa | N-linked glycosylation at Asn118 could alter migration, but a visible shift is unproven. |
| Higher-mass band | Could reflect a homooligomer if the association survives sample preparation. |
| Weak or absent lysate band | Membrane localization may limit recovery in the lysate. |
💡Expected TSPAN8 appearanceUniProt predicts TSPAN8 at 26 kDa; no empirical band size is supplied, and neither a visible Asn118 glycosylation shift nor oligomer migration is established, so confirm any candidate band with antibody identity controls.
| UniProt predicted mass | Sets the unmodified reference near 26 kDa. |
| N-linked glycosylation at Asn118 | Could increase apparent mass if the site carries a glycan; the size change is unknown. |
| Unmodified Asn118 site | Would avoid any mass contribution from that glycan; distinct bands are unproven. |
| Homooligomer formation | Could produce higher-mass species if oligomers survive electrophoresis; their migration is unknown. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-bound TSPAN8 may be poorly recovered. | Check the membrane fraction and improve membrane-protein extraction. |
| Band higher than expected | Asn118 glycosylation or a persistent homooligomer is possible. | Compare glycosidase-treated and thoroughly denatured samples with antibody controls. |
| Broad smear instead of sharp band | Incomplete solubilization of this membrane protein may complicate migration. | Optimize detergent extraction and denaturation, then verify specificity. |
| Multiple bands | Asn118 modification or persistent homooligomers could contribute; band identities are unknown. | Compare glycosidase-treated and denatured samples and check antibody specificity. |
| Weak or no signal | Membrane extraction may yield little TSPAN8. | Enrich the membrane fraction and verify antibody performance with a positive control. |
Experiment evidence
Western blot images showing TSPAN8 band
A06997 · TSPAN8
A06997 · TSPAN8
A06997S129 · TSPAN8
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TSPAN8 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for TSPAN8 in Western blot, you can use appendix tissue, which HPA rates high for TSPAN8 expression.
Positive control: Appendix (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: As a multi-pass membrane protein, TSPAN8 may require effective membrane solubilization for detection.
HPA tissue expression evidence for TSPAN8
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 |
|---|---|---|---|---|
| Appendix | endocrine cells | High | Protein (IHC) | HPA → |
| Colon | endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | endocrine cells | High | Protein (IHC) | HPA → |
| Gallbladder | glandular cells | High | Protein (IHC) | HPA → |
| Kidney | proximal tubules (microvilli) | 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 → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TSPAN8 Western Blot Tips
Deeper troubleshooting and optimisation questions for TSPAN8, answered from its protein features.
How should TSPAN8 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.
Could annotated isoforms explain multiple TSPAN8 bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore provides no annotated isoform assignment for additional bands.
Which TSPAN8 glycosylation site should I consider?
PTM · UniProt lists one N-linked glycosylation site at Asn118, using UniProt sequence numbering. Check the numbering convention before comparing it with an antibody or paper. The site’s presence does not establish how much of the protein is glycosylated in your sample.
Does this guide establish induction of TSPAN8?
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 TSPAN8 Western blot?
Transfer · TSPAN8 is a predicted 26 kDa, 237-residue multi-pass membrane protein. Choose transfer conditions that retain proteins around 26 kDa, and check transfer with a total-protein stain. Adjust transfer conditions if protein remains in the gel or passes through the membrane.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A06997 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 TSPAN8 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Should TSPAN8 run at its predicted 26 kDa?
Interpretation · Its predicted mass is 26 kDa, but no observed band position is supplied. Use 26 kDa as a reference, not a required apparent mass. The listed glycosylation site alone cannot establish a visible shift or explain a mass difference.
TSPAN8 is a cell-membrane, multi-pass protein. Prepare comparable samples with the same membrane-protein extraction conditions, and normalize signals to an appropriate total-protein measure. Differences in extraction can affect the measured signal.
UniProt reports that TSPAN8 forms homooligomers. A higher band could warrant checking whether it persists under different denaturation conditions, but the interaction annotation alone does not establish that a particular band is an oligomer.
Compare them with the predicted 26 kDa mass and assess whether they change with denaturation or sample preparation. UniProt lists Asn118 N-linked glycosylation and homooligomer formation, but neither feature identifies an unexpected band by itself. The supplied record gives no observed band position.
TSPAN8 is a cell-membrane, multi-pass protein. Prepare comparable samples with the same membrane-protein extraction conditions, and normalize signals to an appropriate total-protein measure. Differences in extraction can affect the measured signal.
UniProt reports that TSPAN8 forms homooligomers. A higher band could warrant checking whether it persists under different denaturation conditions, but the interaction annotation alone does not establish that a particular band is an oligomer.
Compare them with the predicted 26 kDa mass and assess whether they change with denaturation or sample preparation. UniProt lists Asn118 N-linked glycosylation and homooligomer formation, but neither feature identifies an unexpected band by itself. The supplied record gives no observed band position.
Boster reagents
TSPAN8 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-Tetraspanin-8 TSPAN8 Antibody
Cat # A06997
Real WB data
Anti-Tetraspanin-8 (S129) TSPAN8 Antibody
Cat # A06997S129
Two the supplier anti-TSPAN8 antibodies have WB images. A06997 lists human and mouse reactivity, with a 1:1000 blot captioned as various cells. A06997S129 lists human, mouse, and rat reactivity; its 1:500 caption names cell lysates, mouse kidney, and rat intestine.
Which to pick: Choose A06997S129 if rat reactivity or a named WB specimen is important. A06997 covers listed human and mouse reactivity, but its WB caption does not identify the cells tested. The named specimens are tested examples, not universal species validation.
Source: BosterBio TSPAN8 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P19075.
- Human Protein Atlas. TSPAN8 tissue expression.
- PMC6929559 — target-verified WB comparison
- PMC7065889 — target-verified WB comparison
- PMC6292129 — target-verified WB comparison