This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
SPTBN1 / Spectrin beta chain, non-erythrocytic 1 · IHC design guide
Design Immunohistochemistry for SPTBN1
Plan chromogenic IHC for SPTBN1 using its observed cytoplasmic and membranous tissue pattern (HPA tissue IHC). Compare staining across cell types with consistent fixation, and check which of its 3 isoforms the antibody recognizes (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SPTBN1 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic and membranous tissue staining (HPA tissue IHC) | |
| Staining pattern | Glandular, ciliated and endothelial cells; cytoplasmic/membranous (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A03164) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Broad tissue expression can limit cell-type contrast (HPA tissue IHC) | |
| Regulation | Regulation not reported (UniProt) | |
| Isoform / epitope | 3 isoforms; verify epitope coverage; no transmembrane segment (UniProt) |
Section 1
Recommended SPTBN1 IHC & IF Protocols
The catalog antibody IHC-P protocol (datasheet A03164) is accompanied by three published SPTBN1 IHC protocols (PMC8017174; PMC6639966; PMC7716908).
| Sample | Paraffin-embedded human Gastric adenocarcinoma tissue; fixative not specified (datasheet A03164) |
| Fixation | Image fixative and duration unreported (datasheet A03164); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A03164); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-SPTBN1, 1:100 - 1:300 (datasheet A03164) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SPTBN1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic and membranous expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 heat retrieval for the catalog antibody (datasheet A03164); citric acid retrieval is reported for a different SPTBN1 antibody (PMC6639966).
Section 2
What Is the Expected SPTBN1 Staining Pattern?
SPTBN1 is a cytoskeletal protein reported in the cytoplasm, cytosol and at the cell membrane; it has no transmembrane segment (UniProt Q01082). In paraffin-section IHC, expect cytoplasmic and membranous staining in several cell populations, including glandular, ciliated and endothelial cells (HPA tissue IHC). HPA rates the tissue staining profile Supported, citing consistency between antibody staining and RNA expression (HPA tissue IHC).
| Cytoplasmic staining, sometimes with a membranous outline, appears in glandular, ciliated or endothelial cells. | This fits HPA’s general cytoplasmic and membranous IHC profile and UniProt’s cytoskeletal and membrane locations (HPA tissue IHC; UniProt Q01082). Read the signal in its tissue and cell context: HPA reports High staining in adrenal gland and appendix glandular cells, bronchial and fallopian-tube ciliated cell bodies, and cerebral-cortex and colon endothelial cells (HPA tissue IHC). |
| Strong staining is confined to nuclei, with little corresponding cytoplasmic signal. | A nuclear-only pattern does not match the supplied SPTBN1 locations or HPA’s tissue profile (UniProt Q01082; HPA tissue IHC). Treat it as suspect; review morphology and the detection controls before scoring it as SPTBN1 (standard IHC practice). |
| An unexpected cell population stains strongly while the expected cells in the same section do not. | HPA describes cell-specific differences, so identify the stained cells before interpreting the result (HPA tissue IHC). If the pattern cannot be reconciled with the reported cells, consider antibody cross-reactivity or endogenous detection activity; use appropriate controls to distinguish these possibilities (standard IHC practice). |
| Color spreads across stroma or much of the section without clear cell boundaries. | Diffuse background is difficult to assign to the cytoplasmic or membranous pattern reported for SPTBN1 (HPA tissue IHC). Check the no-primary control, blocking and washes, and whether chromogen development obscured the cells (standard IHC practice). |
| An apparently intact known-positive cell population shows no detectable signal. | Absence of signal in a population reported as High by HPA, such as adrenal gland glandular cells, raises a run-level concern but does not alone identify its cause (HPA tissue IHC). Check a positive control and the primary-antibody and detection steps before interpreting a test section as negative (standard IHC practice). |
💡Expected SPTBN1 appearanceCall a section positive when recognisable glandular, ciliated cell-body or endothelial cells show appreciable cytoplasmic staining, with possible membranous staining, in an HPA-reported context; nuclear-only or cell-independent color is suspect (HPA tissue IHC; UniProt Q01082).
| Location and topology (UniProt Q01082) | UniProt lists cytoplasm, cytoskeleton, cytosol and cell membrane, with no transmembrane segment. Interpret an apparent membrane outline alongside cytoplasmic staining and cell morphology; the record does not establish a required membrane-only pattern (UniProt Q01082; HPA tissue IHC). |
| Cell and tissue context (HPA tissue IHC) | HPA reports High staining in selected glandular, ciliated and endothelial cells, but Low staining in glial cells of hippocampus and caudate and squamous cells of oral mucosa, esophagus and vagina. Use the named cell population when comparing sections; a low-staining population is a weaker positive reference (HPA tissue IHC). |
| Antibody evidence (HPA antibody validation) | HPA012685 and HPA013149 each have Supported IHC status (HPA antibody validation). That supports the reported IHC patterns; it does not establish that every reagent or staining run will produce the same result (HPA antibody validation; standard IHC practice). |
| Isoforms and epitope coverage (UniProt Q01082) | UniProt lists 3 isoforms and reports isoform 2 protein in brain, lung and kidney (UniProt Q01082). The payload gives no epitope for the antibody used here, so isoform recognition and isoform-specific staining cannot be inferred from this record (UniProt Q01082). |
| IF/ICC Q&A: What pattern is reported? (HPA subcellular) | HPA reports cytosolic ICC-IF localisation but marks its main-location assignment uncertain; its image list includes A-431, U-251MG and U2OS (HPA subcellular). Use that observation as a cautious comparison for IF/ICC, not as a required pattern for chromogenic tissue IHC (HPA subcellular; HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive cells are blank (HPA tissue IHC). | The run may have failed at primary-antibody incubation or detection; a blank section alone cannot distinguish these causes (standard IHC practice). | Inspect a positive control containing an HPA-reported High cell population, then check antibody preparation, incubation, retrieval and detection records (HPA tissue IHC; standard IHC practice). |
| Expected cells stain weakly, but the section has clear morphology (HPA tissue IHC). | HPA staining levels vary by cell population; a reported Low population offers a weak benchmark (HPA tissue IHC). | Compare the same named cell population with an HPA-reported High reference; review routine retrieval and primary-antibody conditions if that reference is also weak (HPA tissue IHC; standard IHC practice). |
| Nuclei dominate the staining (UniProt Q01082; HPA tissue IHC). | The result conflicts with the supplied cytoplasmic and membranous locations; its specific cause is undetermined (UniProt Q01082; HPA tissue IHC). | Check a no-primary control and section morphology; withhold a positive SPTBN1 call until a matching cellular pattern is demonstrated (standard IHC practice; HPA tissue IHC). |
| Broad, hazy chromogen covers cells and tissue spaces (standard IHC practice). | Nonspecific background or detection activity may obscure the reported cellular pattern (standard IHC practice; HPA tissue IHC). | Review the no-primary control, blocking, washes and chromogen development; score only signal that can be assigned to intact cells (standard IHC practice). |
| Unexpected cells stain more strongly than the HPA-reported cells (HPA tissue IHC). | Cross-reactivity or endogenous detection activity is possible; the supplied profile does not identify the unexpected signal (HPA tissue IHC; standard IHC practice). | Identify both cell populations on the counterstained section, compare a no-primary control and, where available, compare an independently validated antibody (standard IHC practice; HPA antibody validation). |
| A membrane-only rim is scored without cytoplasmic context (UniProt Q01082; HPA tissue IHC). | UniProt includes a membrane location, but HPA describes a general cytoplasmic and membranous tissue profile (UniProt Q01082; HPA tissue IHC). | Reassess the cell boundaries and cytoplasm in a reported positive population; document the observed compartment instead of treating a rim alone as definitive (HPA tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing SPTBN1 staining
A03164 · IHCImmunohistochemical analysis of paraffin-embedded human Gastric adenocarcinoma. 1, Antibody was diluted at 1:200(4° overnight). 2, Tris-EDTA
M03164-1 · IHCHuman kidney was stained with anti-SPTBN1 rabbit antibody
M03164-1 · IHCRat kidney was stained with anti-SPTBN1 rabbit antibody
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000115306-SPTBN1/tissue/cerebral+cortex#imid_3487336
HPA · LungHuman Protein Atlas IHC, Lung. Source: https://www.proteinatlas.org/ENSG00000115306-SPTBN1/tissue/lung#imid_3487199
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000115306-SPTBN1/tissue/colon#imid_3487246
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000115306-SPTBN1/tissue/liver#imid_3487263
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA013149; green = SPTBN1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA013149; green = SPTBN1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SPTBN1 IHC & IF
🧪Run bronchus first: ciliated cell bodies should stain strongly (HPA: High in bronchus ciliated cells). HPA detects SPTBN1 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls for the negative comparison, and assess background in acellular luminal spaces and nuclei on the positive slide (HPA: no negative tissues; UniProt Q01082: cytoplasmic and cytoskeletal localization).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: SPTBN1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SPTBN1 in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (uncertain) (HPA subcellular).
Technical controls: Include a secondary-only slide with the primary omitted, a concentration-matched nonimmune antibody of the same host species and isotype as the primary, and SPTBN1-knockout material if available as a biological negative (standard IHC practice). In bronchus, check luminal debris and endogenous peroxidase background against the secondary-only slide (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected A03164 paraffin-section caption does not state a fixative (A03164 tissue-IHC caption). Tris-EDTA at pH 9.0 is a documented retrieval starting point, but the caption does not establish that retrieval is required; frozen sections or IF cannot be judged easier from the supplied evidence (A03164 tissue-IHC caption). For IF/ICC, HPA reports a cytosolic signal of uncertain localization and images in A-431, U-251MG and U2OS; assess autofluorescence with a no-primary control (HPA: subcellular; standard IF practice).
HPA tissue IHC evidence for SPTBN1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — High consistency between antibody staining and RNA expression data.). 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Ciliated cells (cell body) | High | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | High | Protein (IHC) | HPA → |
| Colon | Endothelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: SPTBN1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced SPTBN1 IHC Tips
Troubleshoot SPTBN1 staining in paraffin sections by checking retrieval, compartment, background and cell-specific scoring before interpreting chromogenic signal.
Which retrieval conditions should I try first for weak SPTBN1 staining?
Use heat-mediated Tris-EDTA at pH 9.0 for SPTBN1 paraffin-section IHC (datasheet A03164). The catalog image used this retrieval with primary antibody at 1:200 overnight at 4 °C, so reproduce those conditions when assessing weak staining (datasheet A03164). Compare retrieved sections with a no-primary control and a consistently stained reference section, while keeping section thickness and detection conditions matched (standard IHC practice). If signal remains weak, adjust heating duration in a small series and inspect morphology after each condition; excessive heating can damage tissue and make staining harder to interpret (standard IHC practice).
Could fixation explain inconsistent SPTBN1 staining between paraffin blocks?
The selected SPTBN1 image documents paraffin-embedded tissue but does not state its fixative, so target-specific fixation sensitivity is unknown (datasheet A03164). Record each block’s fixative and fixation duration, then compare blocks with the same retrieval, antibody dilution and chromogenic detection conditions (standard IHC practice). Include tissue with a reproducible staining pattern in each run to distinguish a block-dependent change from a run-wide detection problem (standard IHC practice). If prolonged or variable fixation is suspected, test retrieval conditions systematically while checking morphology; neither the tissue staining profile nor SPTBN1 topology establishes a particular fixation effect (standard IHC practice; HPA tissue IHC; UniProt Q01082 topology).
What staining pattern is plausible for SPTBN1 in tissue sections?
Expect cytoplasmic or membrane-associated staining rather than a predominantly nuclear pattern: SPTBN1 is assigned to the cytoskeleton, cytosol and cell membrane (UniProt Q01082 subcellular location). The tissue atlas describes general cytoplasmic and membranous expression, so score those compartments separately when a section shows both (HPA tissue IHC). SPTBN1 has no transmembrane segment; a membrane-adjacent chromogenic rim can reflect its cytoskeletal association and does not, by itself, establish membrane insertion (UniProt Q01082 topology and subcellular location). Compare staining across intact cells and matched controls before calling a thin edge deposit specific, because section folds and tissue edges can concentrate chromogen (standard IHC practice).
How should isoforms affect my interpretation of uneven SPTBN1 staining?
SPTBN1 has 3 recorded isoforms—Long, Short and 2—so the antibody’s mapped epitope matters when comparing cells or specimens (UniProt Q01082 isoforms; standard IHC practice). Isoform 2 has protein-level evidence in brain, lung and kidney, but that observation alone cannot establish which isoform produced chromogenic staining in a section (UniProt Q01082 tissue specificity; standard IHC practice). Check whether the catalog antibody’s epitope falls within a sequence shared by the isoforms before assigning an isoform-specific interpretation (standard IHC practice). The recorded CH domains at residues 54–158 and 173–278, and PH domain at 2197–2307, provide sequence landmarks, not epitope identification (UniProt Q01082 domains).
How can IF help assess an ambiguous SPTBN1 IHC pattern?
In a separate IF experiment, multiplex SPTBN1 with a marker for the cell type being evaluated so signal can be assigned to cells rather than nearby structures (standard IF practice). Choose fluorophores in channels with low tissue autofluorescence, check each channel separately and include single-stain controls before interpreting overlap (standard IF practice). SPTBN1 is recorded in the cytosol, cytoskeleton and at the cell membrane, with no transmembrane segment; use permeabilisation when the antibody epitope is intracellular and verify the epitope’s accessibility first (UniProt Q01082 subcellular location and topology; standard IF practice). Treat IF localisation as a cross-check of the chromogenic pattern, while evaluating the paraffin-section IHC result on its own controls (standard IHC/IF practice).
How do I distinguish SPTBN1 signal from chromogenic background?
Run a no-primary control and inspect deposits in stroma, damaged areas and section edges before attributing diffuse color to SPTBN1 (standard IHC practice). Use an appropriate peroxidase block for enzyme-based detection, then compare blocking and washing conditions across otherwise matched sections (standard IHC practice). The catalog paraffin image used primary antibody at 1:200 overnight at 4 °C and secondary antibody at 1:200 for 45 minutes at room temperature; these are documented image conditions, not background thresholds (datasheet A03164). Interpret residual cytoplasmic or membranous staining against controls and tissue morphology, since those compartments are plausible for SPTBN1 (HPA tissue IHC; UniProt Q01082 subcellular location; standard IHC practice).
How should I quantify SPTBN1 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and score cytoplasmic and membranous staining separately before comparing specimens (HPA tissue IHC; standard IHC practice). For each population, record the percentage of positive cells and an H-score based on staining intensity; use stained-cell density per mm² when cell abundance itself is the question (standard IHC practice). Normalise cell counts to viable tissue area, apply the same positivity threshold and imaging settings, and exclude folds or necrotic regions consistently (standard IHC practice). Report the compartment and cell denominator alongside each result, because SPTBN1 shows broad tissue expression and a whole-section average can obscure differences between cell populations (HPA tissue IHC; standard IHC practice).
When should I distrust a strong SPTBN1-positive area?
Check whether staining occupies a plausible cytoplasmic or membrane-associated compartment; predominantly nuclear signal needs additional validation for this target (UniProt Q01082 subcellular location; HPA tissue IHC; standard IHC practice). Confirm which cells are stained: high staining is reported in glandular cells of the appendix and endothelial cells of the colon, so neighboring cell populations need separate assessment (HPA tissue IHC). Distrust signal confined to a tissue edge, fold or necrotic area, particularly when comparable intact cells lack staining (standard IHC practice). Compare a no-primary control and an appropriate peroxidase-blocked section to assess nonspecific deposits or endogenous enzyme activity before calling that area SPTBN1-positive (standard IHC practice).
Boster reagents
Best SPTBN1 / Spectrin beta chain, non-erythrocytic 1 IHC Antibodies
Two anti-SPTBN1 antibodies have IHC images from human gastric adenocarcinoma or human and rat kidney (catalog IHC captions). Both list IF and human, mouse and rat reactivity (catalog applications and reactivity).
Real IHC data
Anti-SPTBN1 Antibody
Cat # A03164
Real IHC data
Anti-SPTBN1 Rabbit Monoclonal Antibody
Cat # M03164-1
A03164 will render with IHC of paraffin-embedded human gastric adenocarcinoma and lists IF/ICC (A03164 IHC caption; catalog applications). M03164-1 will render with human kidney IHC; its catalog also documents rat kidney IHC and lists IF (M03164-1 IHC captions; catalog applications).
Which to pick: For paraffin-section IHC, choose A03164: its own image documents that preparation, although the fixative is unreported (A03164 IHC caption). For IF/ICC, choose A03164 when ICC is required (catalog applications); M03164-1 is a monoclonal IF option (catalog clonality and applications). For cross-species tissue IHC, M03164-1 has images from human and rat kidney; both antibodies list human, mouse and rat reactivity, but the M03164-1 captions do not report tissue processing or fixative (M03164-1 IHC captions; catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q01082 (SPTB2_HUMAN, Spectrin beta chain, non-erythrocytic 1).
- Human Protein Atlas. SPTBN1 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. SPTBN1 subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. SPTBN1 antibody validation summary (2 antibodies).
- SPTBN1 Prevents Primary Osteoporosis by Modulating Osteoblasts Proliferation and Differentiation and Blood Vessels Formation in Bone. Frontiers in cell and developmental biology 2021 — PMC8017174.
- Spatial UBE2N protein expression indicates genomic instability in colorectal cancers. BMC cancer 2019 — PMC6639966.
- PRAJA is overexpressed in glioblastoma and contributes to neural precursor development. Genes & cancer 2017 — PMC5620009.
- Co-expression analysis of pancreatic cancer proteome reveals biology and prognostic biomarkers. Cellular oncology (Dordrecht, Netherlands) 2020 — PMC7716908.
- PubMed PMID:1527002 — UniProt-cited evidence.
- PubMed PMID:11665863 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SPTBN1 (Spectrin beta chain, non-erythrocytic 1). Expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A03164). Validated IHC antibody: A03164 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human Gastric adenocarcinoma tissue; fixative not specified (datasheet A03164); 2. Fixation: Image fixative and duration unreported (datasheet A03164); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: Tris-EDTA pH 9.0 (datasheet A03164); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-SPTBN1, 1:100 - 1:300 (datasheet A03164); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Broad tissue expression can limit cell-type contrast (HPA tissue IHC); Isoform / epitope: 3 isoforms; verify epitope coverage; no transmembrane segment (UniProt); Regulation: Regulation not reported (UniProt). Evidence: UniProt Q01082, HPA tissue IHC · Supported, HPA ICC-IF · cytosol., Datasheet A03164, PMC8017174, PMC6639966. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SPTBN1/immunohistochemistry-ihc-guide.