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UBL3 / Ubiquitin-like protein 3 · IHC design guide
Design Immunohistochemistry for UBL3
Plan chromogenic IHC for UBL3 in paraffin sections with the catalog antibody's 1:50–1:200 dilution range (datasheet). Rectal glandular cells show medium staining, but the tissue pattern awaits external verification (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
UBL3 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 staining (HPA tissue IHC); cell membrane (UniProt) | |
| Staining pattern | Cytoplasmic staining in several tissues and endothelial cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Rectum | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Tissue staining awaits external verification (HPA tissue IHC) | |
| Regulation | Regulation not annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; mature chain spans residues 1–114 (UniProt) |
Section 1
Recommended UBL3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published UBL3 immunohistochemistry protocol (PMC10295865).
| Sample | Paraffin-embedded rat testis tissue; fixative not specified (datasheet A13057) |
| Fixation | Image fixative and duration unreported (datasheet A13057); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| 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-UBL3, 1:50-1:200 (datasheet A13057) |
| 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 | UBL3-positive staining in glandular cells of rectum (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in several tissues and in endothelial cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen retrieval); the published protocol does not report retrieval (PMC10295865).
Section 2
What Is the Expected UBL3 Staining Pattern?
UBL3 is annotated at the cell membrane despite having no transmembrane segment (UniProt O95164 topology). In tissue IHC, expect mainly cytoplasmic staining across several tissues, including glandular and endothelial cells (HPA tissue IHC). Rectal glandular cells provide a documented Medium example (HPA tissue IHC). Treat this as a provisional pattern: HPA rates the tissue staining Uncertain, pending external verification (HPA tissue IHC).
| Cytoplasmic chromogen in rectal glandular cells, with distinguishable cell outlines and little surrounding deposit. | This matches the documented Medium rectal glandular staining and the broader cytoplasmic tissue profile (HPA tissue IHC). It is a useful comparison, not proof of antibody specificity: HPA rates its tissue IHC evidence Uncertain (HPA tissue IHC). |
| A strong, exclusively nuclear pattern replaces the expected cytoplasmic signal. | Exclusive nuclear staining conflicts with the tissue IHC profile (HPA tissue IHC) and the cell membrane annotation (UniProt O95164). Consider nonspecific staining or an interpretation error; inspect controls before assigning a new UBL3 location. The approved centrosome finding comes from ICC-IF, a different assay (HPA subcellular). |
| Strong staining appears in adipocytes or bronchial respiratory epithelial cells. | Those cell types were reported as Not detected in the sampled tissues (HPA tissue IHC). Check for cross-reactivity, endogenous detection activity, or misidentified cells with appropriate controls (general IHC practice). A negative HPA observation is a comparison point, not proof that every specimen must be negative. |
| Brown deposit spreads across stroma, empty spaces, or many cell types without clear cellular boundaries. | A diffuse pattern cannot be assigned confidently to the reported cytoplasmic staining in cells (HPA tissue IHC). Examine the no-primary control and reagent background; blocking, washing, and detection chemistry can influence nonspecific deposit in chromogenic IHC (general IHC practice). |
| No signal appears in rectal glandular cells that were selected as the positive comparison. | HPA reports Medium staining in this cell population (HPA tissue IHC), so first check tissue preservation, antibody and detection performance, and whether the glands are present on the section (general IHC practice). Because HPA rates IHC reliability Uncertain, absence alone does not establish UBL3 loss (HPA tissue IHC). |
💡Expected UBL3 appearanceCall an IHC result provisionally positive when rectal glandular cells show discernible, approximately Medium cytoplasmic staining (HPA tissue IHC); widespread featureless deposit or dominant nuclear staining warrants control review rather than a positive call (general IHC practice; HPA tissue IHC).
| Tissue IHC evidence strength | HPA describes cytoplasmic expression in several tissues and endothelial cells but rates the tissue result Uncertain, pending external verification (HPA tissue IHC). Both listed antibodies have Uncertain IHC status (HPA antibodies: HPA053772, HPA058781). Judge weak or unexpected staining conservatively. |
| Compartment and membrane association | UniProt places UBL3 at the cell membrane and lists no transmembrane segment (UniProt O95164 topology). HPA tissue IHC describes cytoplasmic staining (HPA tissue IHC). These annotations support checking cellular localisation, but do not require a sharp membrane rim on a chromogenic section. |
| Tissue and cell context | Rectal glandular cells are Medium, while glandular cells in thyroid, adrenal, stomach, duodenum, and small intestine and lung alveolar cells are Low (HPA tissue IHC). Several other sampled cell populations are Not detected (HPA tissue IHC). Compare like cell types; whole-tissue intensity can obscure that distinction (general IHC practice). |
| Processing and epitope limits | UniProt annotates a 1–114 chain, a 115–117 propeptide, and a cysteine methyl ester at residue 114 (UniProt O95164). No antibody epitope is supplied, so these annotations cannot predict which processed form the IHC-validated antibody detects or whether retrieval changes its signal. |
| IF/ICC Q&A: should a centrosomal signal be expected here? | HPA approves centrosome localisation in ICC-IF and lists images from A-431, MCF-7, U2OS, and NIH 3T3 (HPA subcellular). That is useful context for an IF/ICC result, but it does not establish a centrosomal chromogenic pattern in paraffin tissue or resolve the Uncertain tissue IHC rating (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Rectal glandular cells are blank, including in a comparison section expected to stain. | The antibody, detection reagents, or tissue section may have failed; rectal glandular cells were Medium in HPA's sampled tissue (HPA tissue IHC; general IHC practice). | Confirm that glands are present, inspect counterstaining and run-level controls, then check antibody dilution, detection steps, and the validated assay instructions (general IHC practice). Do not infer a UBL3-specific fixation or retrieval effect from this result. |
| Most cells and extracellular spaces show similar chromogen. | Nonspecific reagent background or endogenous detection activity can obscure a cellular pattern (general IHC practice). The reported tissue pattern is cytoplasmic within cells (HPA tissue IHC). | Review a no-primary control, blocking and washes; assess endogenous activity for the chosen detection chemistry (general IHC practice). Score cellular staining only after background is separable from the tissue pattern. |
| Adipocytes or bronchial respiratory epithelial cells stain strongly. | These cell populations were Not detected in the HPA samples (HPA tissue IHC); cross-reactivity, background, or cell-identification error is possible (general IHC practice). | Verify the cell type on the counterstained section, compare a no-primary control, and seek independent antibody or orthogonal evidence before interpreting the signal as UBL3 (general IHC practice). |
| Only a crisp membrane rim is accepted as positive, so cytoplasmic cells are scored negative. | A membrane-only scoring rule overlooks HPA's cytoplasmic tissue staining (HPA tissue IHC). UniProt's membrane annotation does not prescribe the visual IHC pattern (UniProt O95164 topology). | Score identifiable cytoplasmic staining in the relevant cells against controls and document any membrane emphasis separately (HPA tissue IHC; general IHC practice). |
| A predominantly nuclear or isolated punctate tissue signal is called the expected IHC result. | Neither pattern is established as the tissue IHC profile (HPA tissue IHC). Centrosome localisation is approved in ICC-IF, not tissue IHC (HPA subcellular). | Recheck cell boundaries, counterstain, and control sections; describe the observed compartment without assigning it to UBL3 until independently supported (general IHC practice; HPA tissue IHC). |
| Low-intensity tissue staining varies between sections or readers. | Several listed tissue cell types are Low and tissue IHC reliability is Uncertain (HPA tissue IHC), making a faint result difficult to distinguish from background. | Compare the same cell population across sections, apply a consistent scoring threshold and include an interpretable positive comparison and background control (general IHC practice). Report borderline staining as equivocal rather than forcing a positive call. |
Experiment evidence
IHC & IF images showing UBL3 staining
A13057 · IHCImmunohistochemistry of paraffin-embedded rat testis using UBL3 antibody at dilution of 1:100 .Perform microwave antigen retrieval with 10 m
A13057 · IHCImmunohistochemistry of paraffin-embedded human stomach using UBL3 antibody at dilution of 1:100 .Perform microwave antigen retrieval with 1
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000122042-UBL3/tissue/cerebral+cortex#imid_16224407
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000122042-UBL3/tissue/colon#imid_16223045
HPA · RectumHuman Protein Atlas IHC, Rectum. Source: https://www.proteinatlas.org/ENSG00000122042-UBL3/tissue/rectum#imid_16223005
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000122042-UBL3/tissue/liver#imid_16223038
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA053772; green = UBL3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA053772; green = UBL3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for UBL3 IHC & IF
🧪Run rectum first; its glandular cells should stain at the reported Medium level (HPA: Rectum, glandular cells, Medium). Use adipose tissue as the negative tissue, evaluating adipocytes, which are reported as Not detected (HPA: Adipose tissue, adipocytes, Not detected); on the rectum slide, treat unstained nonglandular cells as internal background references only after confirming their identity and staining status.
Positive control tissue: Rectum (Glandular cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show UBL3 in A-431, MCF-7, U2OS, NIH 3T3, with annotated localisation: Centrosome (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a concentration-matched isotype control appropriate to the primary antibody’s host species and clonality, and UBL3 knockout tissue or a peptide-blocked primary antibody if the matching immunizing peptide is available (standard IHC controls). For chromogenic staining of rectum, quench endogenous peroxidase and assess background from any endogenous biotin if using a biotin-based detection system (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence; the selected paraffin-section caption also leaves its fixative unreported (selected SKU A13057 caption: fixative not stated). The caption specifies microwave retrieval in 10 mM PBS, pH 7.2 for rat testis, but does not establish that retrieval is required for rectum (selected SKU A13057 caption). Paraffin IHC has direct caption evidence, while the supplied IF evidence consists of cell-line images; whether frozen sections or IF are easier for this tissue is unreported (selected SKU A13057 caption; HPA: subcellular ICC-IF images).
HPA tissue IHC evidence for UBL3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Pending external verification. Antibody staining in cells/structures not annotated, view images.). 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 |
|---|---|---|---|---|
| Rectum | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | 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 → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced UBL3 IHC Tips
Troubleshoot UBL3 staining in paraffin sections by checking retrieval, compartment, controls and cell-specific scoring before interpreting chromogenic signal.
How should I retrieve UBL3 when paraffin-section staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). If staining remains weak, compare a matched section using the 10 mM PBS, pH 7.2 microwave retrieval reported for paraffin-embedded rat testis with antibody A13057 (caption A13057). Keep primary antibody, detection and imaging conditions constant across that comparison so retrieval is the variable being tested (standard IHC practice). The caption reports 1:100 antibody dilution for that image, but does not establish an optimal dilution for other specimens (caption A13057). Include a no-primary control to distinguish improved antigen signal from retrieval-related background (standard IHC practice).
Could fixation explain weak UBL3 staining in my paraffin sections?
UBL3-specific sensitivity to fixation is unknown from the supplied evidence, and the selected paraffin-section caption does not state a fixative (caption A13057). Record the actual fixative, fixation duration and processing history for each specimen before comparing staining intensity (standard IHC practice). If matched material is available, compare shorter and longer fixation conditions while holding retrieval, antibody dilution and chromogenic development constant (standard IHC practice). Assess preserved morphology alongside signal, because damaged sections can make apparent staining differences difficult to interpret (standard IHC practice). Do not infer a fixation preference from UBL3 membrane annotation or tissue staining patterns (UniProt O95164; HPA tissue IHC).
Which staining compartment should count as plausible UBL3 signal?
Evaluate membrane-associated and cytoplasmic signal separately: UniProt places UBL3 at the cell membrane, whereas the HPA tissue profile describes cytoplasmic staining in several tissues and endothelial cells (UniProt O95164 subcellular; HPA tissue IHC). UBL3 has no annotated transmembrane segment, so a sharply outlined membrane pattern should not be treated as its only possible appearance (UniProt O95164 topology). The HPA centrosome location comes from subcellular ICC/IF evidence and is a useful comparison, but its tissue-IHC reliability is marked Uncertain (HPA subcellular; HPA tissue IHC). Inspect high magnification fields and score compartments separately, with a no-primary section to assess nonspecific deposits (standard IHC practice).
How might UBL3 processing affect interpretation of an antibody epitope?
Map the antibody immunogen or stated epitope against the 117-aa precursor before interpreting staining (UniProt O95164; standard IHC practice). UniProt annotates a ubiquitin-like domain at residues 10–88, a chain at 1–114 and a 115–117 propeptide, so an epitope near the terminus warrants particular scrutiny (UniProt O95164 processing and domains). Cysteine 114 has an annotated methyl ester, and UBL3 carries membrane-associated modification keywords; epitope accessibility still requires experimental testing for this antibody (UniProt O95164 modified residues and keywords). No isoforms are annotated in the supplied record, so do not explain divergent tissue patterns by asserting a specific splice variant (UniProt O95164 isoforms). Compare retrieval conditions and an independent epitope antibody, if available, using matched sections (standard IHC practice).
How can IF help resolve ambiguous chromogenic UBL3 localisation?
Use IF on a matched specimen to compare UBL3 with a marker for the cell type being evaluated, and inspect colocalisation at single-cell resolution (standard IF practice). Choose fluorophores after checking the specimen's autofluorescence, favouring a channel with clear separation from that background (standard IF practice). Because UBL3 is membrane-associated without an annotated transmembrane segment, select permeabilisation according to the antibody epitope's accessible side of the membrane and validate that choice experimentally (UniProt O95164 topology; standard IF practice). Include single-label and no-primary controls to assess bleed-through and nonspecific fluorescence (standard IF practice). Treat centrosome localisation reported by HPA ICC/IF as context for image review, without assuming that every tissue section will show the same pattern (HPA subcellular).
What should I change when UBL3 IHC has diffuse background?
First inspect the no-primary control and tissue edges to determine whether diffuse colour persists without the UBL3 antibody (standard IHC practice). For peroxidase-based chromogenic detection, check the endogenous peroxidase block and shorten chromogen development if control sections also darken (standard IHC practice). Then titrate the primary antibody and strengthen washing while preserving a matched positive reference section (standard IHC practice). The reported 1:100 dilution belongs to the A13057 rat-testis image and may need adjustment for a different specimen or detection system (caption A13057; standard IHC practice). Judge any improvement against cell morphology and compartment distribution, since the HPA tissue-IHC reliability is Uncertain (HPA tissue IHC).
How should I quantify heterogeneous UBL3 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because HPA reports cytoplasmic tissue staining while UniProt annotates cell-membrane localisation (HPA tissue IHC; UniProt O95164 subcellular). For each predefined region, record the percentage of positive cells and intensity grades, then calculate an H-score from 0–300 if intensity scoring is reproducible (standard IHC practice). For sparse positive structures, report positive-cell density per mm² of evaluable tissue instead (standard IHC practice). Normalise comparisons to the same cell type, tissue area and staining batch, excluding folds, necrosis and section edges by a preset rule (standard IHC practice). Report compartments separately and keep exposure-independent chromogenic thresholds consistent across sections (standard IHC practice).
What would make a UBL3-positive IHC result convincing?
A convincing result has repeatable staining in an anatomically identifiable cell population, appropriate morphology and little signal in the no-primary control (standard IHC practice). Membrane-associated staining is compatible with UniProt, while cytoplasmic tissue staining is reported by HPA; neither pattern alone proves specificity, particularly given HPA's Uncertain tissue-IHC reliability (UniProt O95164 subcellular; HPA tissue IHC). HPA lists medium glandular-cell staining in rectum, which can guide comparison if that tissue is available (HPA tissue IHC). Discount colour concentrated at section edges, necrotic regions or sites with endogenous enzyme activity before scoring (standard IHC practice). Recheck an unexpected cell type or isolated compartment with matched controls and, where feasible, an independent antibody epitope (standard IHC practice).
Boster reagents
Best UBL3 / Ubiquitin-like protein 3 IHC Antibodies
A13057 has IHC images of paraffin-embedded rat testis, human stomach, and mouse brain (catalog IHC image captions). No IF image is supplied (catalog: IF images).
Real IHC data
Anti-Ubiquitin-like protein 3 UBL3 Antibody
Cat # A13057
A13057 is listed for IHC and reacts with human, mouse, and rat samples (catalog: applications and reactivity). Its IHC images show paraffin-embedded rat testis, human stomach, and mouse brain (catalog IHC image captions).
Which to pick: For tissue IHC, choose A13057; its own captions document paraffin sections stained at 1:100 after microwave retrieval in 10 mM PBS at pH 7.2, but do not report the fixative (catalog IHC image captions). No SKU can be recommended as IF/ICC validated because A13057 has no listed IF/ICC application or IF image (catalog: applications and IF images). For cross-species IHC, A13057 lists human, mouse, and rat reactivity; its clone identity is unreported (catalog: reactivity and clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O95164 (UBL3_HUMAN, Ubiquitin-like protein 3).
- Human Protein Atlas. UBL3 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. UBL3 subcellular location (ICC-IF): Localized to the centrosome..
- Human Protein Atlas. UBL3 antibody validation summary (2 antibodies).
- UBL3 Interacts with PolyQ-Expanded Huntingtin Fragments and Modifies Their Intracellular Sorting. Neurology international 2024 — PMC11503352.
- UBL3 Interacts with Alpha-Synuclein in Cells and the Interaction Is Downregulated by the EGFR Pathway Inhibitor Osimertinib. Biomedicines 2023 — PMC10295865.
- Identification of a potential tumor suppressor gene, UBL3, in non-small cell lung cancer. Cancer biology & medicine 2020 — PMC7142850.
- Bone Mesenchymal Stromal Cell-Derived Extracellular Vesicles Protect Articular Cartilage Through Regulating tRF-Gln-TTG-019/UBL3. Mediators of inflammation 2025 — PMC12181665.
- PubMed PMID:10375635 — UniProt-cited evidence.
- PubMed PMID:11230166 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for UBL3 (Ubiquitin-like protein 3). Expected localisation Cytoplasmic staining (HPA tissue IHC); cell membrane (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A13057 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Rectum (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded rat testis tissue; fixative not specified (datasheet A13057); 2. Fixation: Image fixative and duration unreported (datasheet A13057); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 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-UBL3, 1:50-1:200 (datasheet A13057); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Tissue staining awaits external verification (HPA tissue IHC); Isoform / epitope: No isoforms annotated; mature chain spans residues 1–114 (UniProt); Regulation: Regulation not annotated (UniProt). Evidence: UniProt O95164, HPA tissue IHC · Uncertain, HPA ICC-IF · centrosome., Datasheet A13057, PMC10295865. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/UBL3/immunohistochemistry-ihc-guide.