This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
UBA6 / Ubiquitin-like modifier-activating enzyme 6 · IHC design guide
Design Immunohistochemistry for UBA6
Plan chromogenic UBA6 IHC in paraffin sections with the catalog antibody at 2–5 μg/ml (datasheet A03213-2). Use the cytoplasmic spermatozoa profile and high staining in elongated or late spermatids as reference points, with caution because staining and RNA expression show very low consistency (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
UBA6 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 in spermatozoa (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic spermatozoa; high in late spermatids (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03213-2) | |
| Positive control | Testis | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent. (standard IHC practice; not target-specific) | |
| Caveat | Staining has very low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Isoform 2: adult > fetal testis (UniProt) | |
| Isoform / epitope | 4 isoforms; check epitope coverage before isoform claims (UniProt) |
Section 1
Recommended UBA6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet A03213-2) is accompanied by published UBA6 IHC protocols for tumour sections (PMC9478149) and human breast tissue microarrays (PMC5675648).
| Sample | Paraffin-embedded human cervical cancer tissue; fixative not specified (datasheet A03213-2) |
| Fixation | Image fixative and duration unreported (datasheet A03213-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A03213-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03213-2) |
| Primary antibody | Rabbit anti-UBA6, 2-5 μg/ml (datasheet A03213-2) |
| Primary incubation | Overnight at 4 °C (datasheet A03213-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03213-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | UBA6-positive staining in elongated or late spermatids of testis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in spermatozoa. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A03213-2). The breast tissue microarray protocol used low-pH retrieval with a different antibody (PMC5675648).
Section 2
What Is the Expected UBA6 Staining Pattern?
In paraffin-section IHC, expect strong cytoplasmic staining in elongated or late spermatids in testis; HPA describes cytoplasmic expression in spermatozoa (HPA tissue IHC: High in elongated or late spermatids). UBA6 has no transmembrane segment (UniProt A0AVT1 topology). Treat the pattern as a guide, because HPA rates the tissue staining Approved while reporting very low consistency with RNA expression and pending external verification (HPA tissue IHC: reliability).
| Strong cytoplasmic signal follows elongated or late spermatids in testis, with identifiable cell boundaries and limited background. | This matches the clearest reported tissue positive: High staining in elongated or late spermatids and a cytoplasmic spermatozoa profile (HPA tissue IHC). Assess the stained cells against section morphology and the counterstain (general IHC practice). |
| Nuclei alone stain strongly in elongated or late spermatids, while their cytoplasm is unstained. | A nuclear-only result conflicts with the reported testis IHC profile (HPA tissue IHC: cytoplasmic expression in spermatozoa). Review morphology and controls before calling it an artifact: HPA also reports nucleoplasm and cytosol in ICC-IF, a different preparation (HPA subcellular). |
| The strongest signal is in adipocytes or adrenal glandular cells rather than late spermatids. | Those cell types were Not detected in the supplied tissue IHC observations (HPA tissue IHC: adipose tissue; adrenal gland). Consider cross-reactivity or detection-system activity, and check whether signal persists in a no-primary control (general IHC practice). |
| Brown signal spreads across tissue, empty spaces, and section edges without following cell morphology. | This is a background pattern rather than the reported cytoplasmic spermatid pattern (HPA tissue IHC). Inspect the no-primary control and review blocking, washes, chromogen development, and section handling as general chromogenic IHC checks (general IHC practice). |
| No specific signal appears in an otherwise intact testis section containing elongated or late spermatids. | That misses the reported High-positive cell population (HPA tissue IHC: testis). Before interpreting absence as biology, check that the relevant cells are present and review antibody, retrieval, detection, and counterstain conditions against the run controls (general IHC practice). |
💡Expected UBA6 appearanceCall a positive result when elongated or late spermatids show High cytoplasmic signal (HPA tissue IHC: testis); isolated nuclear staining or signal spread across unrelated cells is discordant with that tissue pattern and needs control review (HPA tissue IHC; general IHC practice).
| Which tissue and cells anchor interpretation? | Testis elongated or late spermatids provide the supplied High-positive IHC reference (HPA tissue IHC). HPA reports Not detected in several sampled cell types, including adipocytes and adrenal glandular cells; these are observed comparisons, not proof that every cell in those tissues lacks UBA6 (HPA tissue IHC). |
| How much weight should the tissue profile carry? | HPA labels its tissue IHC reliability Approved, yet reports very low consistency between staining and RNA expression, with external verification pending (HPA tissue IHC: reliability). Interpret discordant fields cautiously and retain tissue morphology and run controls in the assessment (general IHC practice). |
| Can the IF result look nuclear? | Yes. HPA ICC-IF reports enhanced nucleoplasm and cytosol localization, with images from U-251MG and HeLa BAC 5376 (HPA subcellular). That supports an IF localization discussion, but it does not replace the reported cytoplasmic spermatozoa pattern for testis IHC (HPA tissue IHC). |
| Could an isoform affect tissue comparisons? | UBA6 has four annotated isoforms; isoform 2 is predominantly expressed in testis and is higher in adult than fetal testis (UniProt A0AVT1: isoforms; tissue specificity). The supplied record does not establish which isoform produced the HPA stain, so do not assign the spermatid signal to isoform 2. |
| What do topology and antibody validation establish? | UBA6 has no signal peptide, propeptide, or transmembrane segment in the supplied record (UniProt A0AVT1: processing; topology). HPA037001 is Approved for IHC and Enhanced for ICC, but those ratings do not identify its epitope or establish retrieval or fixation sensitivity (HPA antibodies: HPA037001). |
| Situation | Likely cause | Next action |
|---|---|---|
| Testis appears negative. | The section may lack identifiable elongated or late spermatids, the reported High-positive cells (HPA tissue IHC: testis). A failed staining run is another possibility (general IHC practice). | Confirm cell-stage morphology, then check the run's positive control and the antibody and detection steps (general IHC practice). Do not score an unrepresented cell population as negative. |
| Only spermatid nuclei stain. | This differs from cytoplasmic spermatozoa staining in tissue IHC (HPA tissue IHC). Nucleoplasm localization in ICC-IF means nuclear signal alone is insufficient to identify the cause (HPA subcellular). | Recheck cell boundaries with the counterstain and compare the no-primary control (general IHC practice); report the compartment discordance rather than treating the ICC-IF result as tissue-IHC confirmation. |
| Adipocytes or adrenal glandular cells stain strongly. | Those sampled cell types were Not detected by HPA tissue IHC (HPA tissue IHC). Cross-reactivity or detection-system activity could explain an unexpected signal (general IHC practice). | Compare with the no-primary control and review staining in the reported testis-positive cells in the same run (HPA tissue IHC: testis; general IHC practice). |
| The whole section has diffuse chromogen. | Signal without cell boundaries does not match the reported cytoplasmic spermatid pattern (HPA tissue IHC). Background from blocking, washing, or detection is possible (general IHC practice). | Inspect the no-primary control; review blocking, wash steps, and chromogen development, then rescore only morphology-linked staining (general IHC practice). |
| IF shows cytosol and nucleoplasm while IHC shows cytoplasmic spermatids. | These observations come from different preparations and sampled cells: ICC-IF reports nucleoplasm and cytosol; testis tissue IHC reports cytoplasmic spermatozoa (HPA subcellular; HPA tissue IHC). | Keep the interpretation tied to each preparation's cells and compartment; use the separate IF/ICC guide for IF workflow (HPA subcellular; HPA tissue IHC). |
| A weak or variable tissue result conflicts with RNA expectations. | HPA reports low tissue RNA specificity and very low consistency between antibody staining and RNA expression (HPA tissue IHC: RNA specificity; reliability). | Describe the observed cell-level stain and controls, and flag the HPA reliability limitation; avoid inferring a validated protein-negative tissue from RNA alone (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing UBA6 staining
A03213-2 · IHCIHC analysis of UBA6 using anti-UBA6 antibody (A03213-2). UBA6 was detected in a paraffin-embedded section of human cervical cancer tissue.
A03213-2 · IHCIHC analysis of UBA6 using anti-UBA6 antibody (A03213-2). UBA6 was detected in a paraffin-embedded section of human lung squamous cell carci
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000033178-UBA6/tissue/cerebral+cortex#imid_9334620
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000033178-UBA6/tissue/colon#imid_9285566
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000033178-UBA6/tissue/liver#imid_9285567
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000033178-UBA6/tissue/kidney#imid_9285560
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody HPA037001; green = UBA6, blue = nucleus, red = microtubules.
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody HPA037001; green = UBA6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for UBA6 IHC & IF
🧪Run testis first and require staining in elongated or late spermatids (HPA: High in elongated or late spermatids). Run adipose tissue as a negative comparator, assessing adipocytes (HPA: Not detected in adipocytes); cells outside the spermatid compartment on the testis slide should show less signal, but their UBA6-negative status is not established by the supplied HPA row.
Positive control tissue: Testis (Elongated or late spermatids, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show UBA6 in U-251MG, HeLa BAC 5376, with annotated localisation: Nucleoplasm (enhanced), Cytosol (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit isotype control for the rabbit catalog antibody, and UBA6-knockout material as a biological negative (selected-SKU caption: rabbit anti-UBA6 antibody; standard IHC practice). Quench endogenous peroxidase and check background in the testis section before interpreting DAB signal (selected-SKU caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative not stated). Heat retrieval in EDTA at pH 8.0 was used for the catalog antibody, but retrieval dependence was not established (selected-SKU caption: EDTA heat retrieval); the supplied evidence does not establish that frozen sections or IF are easier. In testis, distinguish signal in the small elongated or late spermatids from counterstain and background by cell morphology and the control slides (HPA: High in elongated or late spermatids; standard IHC practice).
HPA tissue IHC evidence for UBA6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Very low consistency between antibody staining and RNA expression data. External characterization data supports antibody staining. Pending external verification.). 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 |
|---|---|---|---|---|
| Testis | Elongated or late spermatids | High | Protein (IHC) | HPA → |
Undetected expression · recommended 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 UBA6 IHC Tips
Troubleshoot UBA6 staining in paraffin sections by checking retrieval, tissue preservation, compartment pattern and controls before comparing signal between samples.
What retrieval should I try first when UBA6 staining is weak?
Start with heat mediated antigen retrieval in EDTA, pH 8.0 (datasheet A03213-2). The selected paraffin section image used that retrieval before incubation with 2 μg/ml antibody overnight at 4°C, so reproduce those conditions when assessing weak staining (datasheet A03213-2). Keep section thickness, heating time and cooling conditions consistent across the comparison, and include a testis control with elongated or late spermatids (standard IHC practice; HPA: High in elongated or late spermatids). If signal remains weak, compare a second retrieval condition on adjacent sections while holding antibody concentration and detection constant; treat any improvement as an assay result requiring specificity checks (standard IHC practice).
Could fixation explain weak or uneven UBA6 staining?
Target specific sensitivity of UBA6 staining to fixation is unknown: the selected paraffin section caption does not report a fixative (datasheet A03213-2). Record each specimen’s fixative and fixation duration, then compare sections processed together using the documented EDTA, pH 8.0 retrieval condition (standard IHC practice; datasheet A03213-2). Use a section from testis containing elongated or late spermatids as a staining control, while recognising that its expected signal does not establish fixation tolerance (HPA: High in elongated or late spermatids; standard IHC practice). If staining differs between processing batches, test matched sections before attributing the difference to UBA6 expression (standard IHC practice).
How should I assess an unexpected UBA6 staining pattern?
Assess chromogenic signal by cell type and compartment, and compare it with the matched negative control (standard IHC practice). Cytoplasmic staining in spermatozoa is reported in tissue IHC, whereas ICC/IF reports UBA6 mainly in nucleoplasm and cytosol; those observations support checking both compartments without requiring identical patterns across preparations (HPA tissue IHC; HPA subcellular). UBA6 has no annotated transmembrane segment, so a crisp membrane only rim warrants a specificity check rather than immediate biological interpretation (UniProt A0AVT1 topology; standard IHC practice). Repeat the comparison using the documented EDTA, pH 8.0 retrieval and review adjacent morphology before scoring a discordant pattern (datasheet A03213-2; standard IHC practice).
Can this stain distinguish UBA6 isoforms or a modified epitope?
Do not assign a positive chromogenic stain to one isoform unless the antibody’s epitope and isoform coverage have been established (standard IHC practice). UBA6 has 4 annotated isoforms, and isoform 2 is predominantly expressed in testis, with higher expression in adult than fetal testis (UniProt A0AVT1). The record also lists modified residues, including phosphothreonine 54 and phosphoserine 301; their presence alone does not establish an effect on this antibody’s binding (UniProt A0AVT1; standard IHC practice). For an isoform or modification claim, obtain epitope mapping and an appropriate independent validation result before interpreting staining differences as molecular specificity (standard IHC practice).
How can IF help check the UBA6 IHC pattern?
Use IF/ICC as a complementary compartment check: reported UBA6 signal is mainly nucleoplasmic and cytosolic (HPA subcellular). For tissue multiplexing, pair UBA6 with a validated marker for elongated or late spermatids, the testis cell population with high reported tissue IHC staining (HPA tissue IHC; standard IF practice). Select fluorophores after inspecting unstained tissue for autofluorescence, and separate their emission from the marker and counterstain channels (standard IF practice). Because the compartments of interest are intracellular and UBA6 has no annotated transmembrane segment, choose permeabilisation compatible with the antibody’s epitope, then compare permeabilised and control preparations rather than assuming the IHC retrieval conditions transfer to IF (HPA subcellular; UniProt A0AVT1 topology; standard IF practice).
How do I separate UBA6 signal from DAB background?
Inspect a section processed without primary antibody to identify detection related staining, and compare it with the full stain at the same development endpoint (standard IHC practice). The selected paraffin section used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, a peroxidase conjugated secondary for 30 minutes at 37°C, and DAB detection (datasheet A03213-2). Include a peroxidase block before detection, and review wash and DAB development conditions if diffuse brown signal persists (standard IHC practice). Give particular scrutiny to edge staining, necrotic regions and pigment before calling individual cells positive (standard IHC practice).
How should I quantify UBA6 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and compartment before measuring signal, using tissue morphology and the expected testis spermatid pattern where relevant (standard IHC practice; HPA tissue IHC). For cellular staining, report the percentage of positive cells and an H-score from 0–300 when intensity grading is reproducible; for sparse positive populations, report positive cells per mm² (standard IHC practice). Normalise counts to the relevant viable cell population or viable tissue area, and apply the same threshold, counterstain review and exclusion rules to every section (standard IHC practice). Analyse processing batches separately or include a shared control, because the selected caption specifies retrieval and antibody conditions but does not report fixation (datasheet A03213-2; standard IHC practice).
What makes a UBA6 positive stain convincing?
A convincing call requires signal in intact cells that exceeds the matched detection control and follows a reproducible compartment and cell type pattern (standard IHC practice). High staining in elongated or late spermatids and cytoplasmic expression in spermatozoa are reported tissue IHC observations; ICC/IF also reports nucleoplasmic and cytosolic localisation (HPA tissue IHC; HPA subcellular). Treat isolated membrane only signal, section edge staining, necrosis and staining present without primary antibody as reasons to investigate artefact or endogenous enzyme activity before assigning UBA6 positivity (UniProt A0AVT1 topology; standard IHC practice). Interpret negative tissue cautiously because HPA rates the tissue staining Approved but reports very low consistency with RNA expression and pending external verification (HPA tissue IHC).
Boster reagents
Best UBA6 / Ubiquitin-like modifier-activating enzyme 6 IHC Antibodies
A03213-2 has real IHC data from human paraffin sections (IHC image captions). Its listed reactivity includes human, monkey, mouse, and rat (catalog reactivity); no IF/ICC data are supplied (catalog applications and images).
Real IHC data
Anti-UBA6 Antibody ®
Cat # A03213-2
A03213-2 is listed for IHC and has images from paraffin sections of human cervical cancer, lung squamous cell carcinoma, and ovarian cancer (catalog applications; IHC image captions). It lists human, monkey, mouse, and rat reactivity, with IHC dilution specified for human tissue only (catalog reactivity; dilution data).
Which to pick: Choose A03213-2 for human paraffin-section IHC: its own captions show staining at 2 μg/ml after EDTA pH 8.0 retrieval; the fixative is unreported (IHC image captions). No supplied SKU is validated for IF/ICC, so there is no IF/ICC pick (catalog applications and images). A03213-2 lists monkey, mouse, and rat reactivity, but the supplied IHC evidence is limited to human sections; assess those species separately before tissue IHC use (catalog reactivity; IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry A0AVT1 (UBA6_HUMAN, Ubiquitin-like modifier-activating enzyme 6).
- Human Protein Atlas. UBA6 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. UBA6 subcellular location (ICC-IF): Mainly localized to the nucleoplasm and cytosol..
- Human Protein Atlas. UBA6 antibody validation summary (1 antibodies).
- Inhibition of UBA6 by inosine augments tumour immunogenicity and responses. Nature communications 2022 — PMC9478149.
- The non-canonical ubiquitin activating enzyme UBA6 suppresses epithelial-mesenchymal transition of mammary epithelial cells. Oncotarget 2017 — PMC5675648.
- UBA6 serves as a prognostic biomarker and promotes tumor progression in pancreatic ductal adenocarcinoma. Translational cancer research 2026 — PMC13190965.
- Altered social behavior and neuronal development in mice lacking the Uba6-Use1 ubiquitin transfer system. Molecular cell 2013 — PMC3640669.
- PubMed PMID:15202508 — UniProt-cited evidence.
- PubMed PMID:17597759 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for UBA6 (Ubiquitin-like modifier-activating enzyme 6). Expected localisation Cytoplasmic staining in spermatozoa (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03213-2). Validated IHC antibody: A03213-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Elongated or late spermatids); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human cervical cancer tissue; fixative not specified (datasheet A03213-2); 2. Fixation: Image fixative and duration unreported (datasheet A03213-2); 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: EDTA pH 8.0 (datasheet A03213-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03213-2); 8. Primary antibody: Rabbit anti-UBA6, 2-5 μg/ml (datasheet A03213-2); 9. Primary incubation: Overnight at 4 °C (datasheet A03213-2). Watch for: Fixation: Keep paraffin-section fixation consistent. (standard IHC practice; not target-specific); Caveat: Staining has very low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 4 isoforms ; check epitope coverage before isoform claims (UniProt); Regulation: Isoform 2: adult > fetal testis (UniProt). Evidence: UniProt A0AVT1, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm and cytosol., Datasheet A03213-2, PMC9478149, PMC5675648. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/UBA6/immunohistochemistry-ihc-guide.