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UBA3 / NEDD8-activating enzyme E1 catalytic subunit · IHC design guide
Design Immunohistochemistry for UBA3
Plan UBA3 IHC in paraffin sections using the observed nuclear and cytoplasmic tissue pattern (HPA tissue IHC). Use testis pachytene spermatocytes as a high staining reference (HPA tissue IHC), and interpret weak or absent staining by cell type.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
UBA3 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 | Nuclear and cytoplasmic staining (HPA tissue IHC) | |
| Staining pattern | Broad nuclear and cytoplasmic staining; high in pachytene spermatocytes (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9838) | |
| Positive control | Testis+4 more · see all | |
| Negative control | Ovary |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9838) | |
| Caveat | Ovarian stroma is unstained despite broad tissue expression (HPA tissue IHC) | |
| Regulation | Ubiquitously expressed (UniProt) | |
| Isoform / epitope | 2 isoforms; check epitope coverage before comparing staining (UniProt) |
Section 1
Recommended UBA3 IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 heat retrieval (datasheet PB9838). The published ICC protocol below uses microwave EDTA retrieval (PMC10984854).
| Sample | Paraffin-embedded mouse testis tissue; fixative not specified (datasheet PB9838) |
| Fixation | Image fixative and duration unreported (datasheet PB9838); 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 PB9838); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9838) |
| Primary antibody | Rabbit anti-UBA3, 0.5-1μg/ml (datasheet PB9838) |
| Primary incubation | Overnight at 4 °C (datasheet PB9838) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9838) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | UBA3-positive staining in pachytene spermatocytes of testis (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear and cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet PB9838); the published microwave EDTA method offers a comparison for optimization (PMC10984854).
Section 2
What Is the Expected UBA3 Staining Pattern?
In paraffin IHC, expect widespread nuclear and cytoplasmic UBA3 staining, with strong staining in testicular pachytene spermatocytes and moderate staining in several other cell types (HPA: tissue IHC). HPA rates the tissue pattern Supported, with medium consistency between staining and RNA data (HPA: reliability). UBA3 has no annotated transmembrane segment; UniProt does not assign it a subcellular location (UniProt Q8TBC4: topology and subcellular annotation).
| Nuclear and cytoplasmic staining appears in pachytene spermatocytes, with staining also present across other tissue cell types. | This fits the ubiquitous IHC profile; pachytene spermatocytes are rated High (HPA: tissue IHC). Compare compartments and cell identity when scoring, since intensity varies by cell type. |
| A structure shows staining only outside cells or within lumens, without the expected intracellular pattern. | That distribution warrants an artefact check against HPA's nuclear and cytoplasmic IHC profile (HPA: tissue IHC). Surface staining alone is less decisive: plasma membrane localisation is approved in ICC-IF (HPA: subcellular). |
| Strong signal occurs in ovarian stroma while a known positive cell population stains weakly or not at all. | HPA reports ovarian stroma as Not detected and testicular pachytene spermatocytes as High (HPA: tissue IHC). Check cell identification, cross-reactivity and endogenous detection activity before assigning the stromal signal to UBA3. |
| Chromogen covers tissue broadly, obscures nuclei or appears in the no-primary control. | Diffuse signal cannot establish UBA3 localisation (general IHC practice). Review blocking, antibody concentration, washing and endogenous enzyme controls; use the HPA cell and compartment pattern to judge whether specific staining remains. |
| No signal appears in a section containing identifiable pachytene spermatocytes. | That conflicts with their High HPA staining level (HPA: tissue IHC). First confirm the cell population and section quality, then check primary antibody use, retrieval and detection with appropriate controls (general IHC practice). |
💡Expected UBA3 appearanceCall positive paraffin IHC when intracellular nuclear and cytoplasmic staining follows the tissue cell pattern, especially High staining in pachytene spermatocytes (HPA: tissue IHC); diffuse deposit or signal confined to empty spaces calls for control review (general IHC practice).
| Tissue and cell type | UBA3 is ubiquitously expressed (UniProt Q8TBC4: tissue specificity), yet HPA grades individual cells from High to Not detected (HPA: tissue IHC). Score identified cells rather than treating every unstained cell as assay failure. |
| IHC evidence strength | The tissue pattern is Supported, with medium staining–RNA consistency (HPA: reliability). HPA034873, HPA034874 and HPA065335 each have Supported IHC status (HPA: antibodies); that status is not an Enhanced designation. |
| Compartment by application | Tissue IHC is described as nuclear and cytoplasmic (HPA: tissue IHC). ICC-IF instead approves plasma membrane and cytosol, with additional nucleoplasm and centrosome localisation (HPA: subcellular); interpret each assay against its own evidence. |
| IF/ICC Q: What localisation should I expect? | A: Mainly plasma membrane and cytosol, with additional nucleoplasm and centrosome signal (HPA: subcellular). The IF/ICC workflow belongs in its separate guide; these observations do not define a paraffin IHC protocol. |
| Protein topology and processing | UBA3 has no transmembrane segment or signal peptide, and its annotated chain spans residues 2–463 (UniProt Q8TBC4: topology and processing). These annotations do not establish fixation sensitivity or a required retrieval condition. |
| Isoform interpretation | UniProt lists two UBA3 isoforms (UniProt Q8TBC4: isoforms). Without an antibody epitope or isoform-specific staining result in these sources, assign the observed pattern to UBA3 without claiming which isoform produced it. |
| Situation | Likely cause | Next action |
|---|---|---|
| Testis is faint or blank. | Pachytene spermatocytes may be absent from the examined area, or the IHC workflow may have failed (HPA: High in pachytene spermatocytes; general IHC practice). | Verify cell identity, inspect a known positive section, and check primary antibody, general retrieval settings and detection controls before interpreting a negative result. |
| Ovarian stroma stains strongly. | This differs from HPA's Not detected observation for ovarian stroma; nonspecific binding or endogenous detection activity is possible (HPA: tissue IHC; general IHC practice). | Compare a no-primary control, assess endogenous enzyme blocking and confirm the cells scored as stroma; retain uncertainty if controls do not resolve the signal. |
| Background obscures cellular boundaries. | Excess antibody, inadequate blocking or insufficient washing can produce broad chromogen deposition (general IHC practice). | Review the no-primary control and optimize antibody concentration, blocking and washes; rescore only where nuclei and cytoplasm remain distinguishable. |
| Only surface staining is visible in paraffin IHC. | HPA's IHC summary emphasizes nuclear and cytoplasmic expression, while membrane localisation is approved in ICC-IF (HPA: tissue IHC; HPA: subcellular). | Compare intracellular staining in known positive cells and examine controls. Do not reject a surface signal solely because it differs from the IHC summary. |
| Staining varies sharply between cell types. | HPA reports High, Medium, Low and Not detected levels across specified cells despite a ubiquitous overall profile (HPA: tissue IHC). | Score identified cell types separately and compare them with the corresponding HPA observations; avoid calling the entire section positive or negative from one population. |
| A claim of isoform-specific or fixation-sensitive staining is needed. | Two isoforms are listed, but the supplied UniProt and HPA records give no isoform-specific IHC pattern or target-specific fixation effect (UniProt Q8TBC4: isoforms; HPA: tissue IHC). | Report the observed UBA3 pattern and the assay conditions without assigning an isoform or a fixation mechanism from these sources. |
Experiment evidence
IHC & IF images showing UBA3 staining
PB9838 · IHCIHC analysis of UBE1C using anti-UBE1C antibody (PB9838). UBE1C was detected in a paraffin-embedded section of mouse testis tissue. Heat med
PB9838 · IHCIHC analysis of UBE1C using anti-UBE1C antibody (PB9838). UBE1C was detected in a paraffin-embedded section of rat testis tissue. Heat media
PB9838 · IFIF analysis of UBE1C using anti-UBE1C antibody (PB9838). UBE1C was detected in immunocytochemical section of A431 cell. Enzyme antigen retri
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000144744-UBA3/tissue/cerebral+cortex#imid_19461875
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000144744-UBA3/tissue/colon#imid_19447613
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000144744-UBA3/tissue/liver#imid_19458446
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000144744-UBA3/tissue/kidney#imid_19447615
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA034873; green = UBA3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA034873; green = UBA3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for UBA3 IHC & IF
🧪Run testis first and confirm staining in pachytene spermatocytes (HPA: High in testis pachytene spermatocytes). Run ovary as the negative tissue and assess its stromal cells (HPA: Not detected in ovarian stroma cells); on the testis slide, use only morphologically identifiable cells that remain unstained as internal negative comparators, without assuming a particular cell type is negative.
Positive control tissue: Testis (Pachytene spermatocytes, HPA High)
Negative control tissue: Ovary (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show UBA3 in A-431, U-251MG, SiHa, U2OS, with annotated localisation: Plasma membrane (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary secondary-only control, a concentration-matched nonimmune rabbit IgG isotype control matched to the primary antibody’s clonality when known, and UBA3 knockout tissue or a validated immunizing-peptide block as a biological specificity control (selected PB9838 caption: rabbit primary; standard IHC practice). For testis, quench endogenous peroxidase and check endogenous biotin background when using the caption’s SABC–DAB detection (selected PB9838 caption: SABC–DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected PB9838 testis caption does not state a fixative (selected PB9838 caption: fixative unreported). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (selected PB9838 caption: EDTA heat retrieval); the supplied evidence does not establish that frozen sections or tissue IF are easier, although HPA reports ICC–IF images in cultured cells (HPA: ICC–IF cell-line images). In testis, score staining specifically in pachytene spermatocytes and assess background in other cells, given the reported cell-specific high signal and the caption’s biotin-based chromogenic detection (HPA: High in pachytene spermatocytes; selected PB9838 caption: SABC–DAB).
HPA tissue IHC evidence for UBA3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium 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 |
|---|---|---|---|---|
| Testis | Pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced UBA3 IHC Tips
Troubleshoot UBA3 staining in paraffin sections by checking retrieval, compartment, controls and scoring against the available tissue evidence.
How should I adjust retrieval when UBA3 staining is weak in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PB9838). Compare matched sections heated for 10 and 20 minutes, then cool them consistently before applying the same antibody concentration and detection steps (standard IHC practice). The illustrated PB9838 tissue section used 1 μg/ml antibody overnight at 4°C, so keep those conditions fixed during the retrieval comparison (caption PB9838). If staining remains weak, test a different retrieval buffer on additional sections as a fallback, recording both signal and tissue preservation (standard IHC practice). Judge the result against expected testis staining and a no-primary control, rather than staining intensity alone (HPA tissue IHC; standard IHC practice).
Could fixation explain variable UBA3 staining between paraffin blocks?
Target-specific fixation sensitivity is unknown for UBA3 in this IHC workflow; the PB9838 caption describes a paraffin section but does not state its fixative (caption PB9838). Record each block’s fixative and fixation duration before comparing staining, and process matched sections together when possible (standard IHC practice). Keep EDTA retrieval at pH 8.0 and the primary antibody at 1 μg/ml constant while assessing those blocks (datasheet PB9838; caption PB9838). Review morphology and the no-primary control alongside signal, because damaged tissue or detection background can confound the comparison (standard IHC practice). Do not infer a UBA3-specific fixation effect from its tissue distribution, topology or modified residue (HPA tissue IHC; UniProt Q8TBC4; caption PB9838).
Should a strong UBA3 stain be nuclear, cytoplasmic or membranous?
Score nuclear and cytoplasmic staining separately in paraffin sections: the tissue IHC profile reports both compartments across tissues (HPA tissue IHC). In cell imaging, the main reported locations are plasma membrane and cytosol, with additional nucleoplasm and centrosome staining (HPA subcellular). UBA3 has no annotated transmembrane segment, so a membrane outline alone does not establish that the protein spans the membrane (UniProt Q8TBC4 topology). Compare compartment patterns within intact cells and across matched sections, using a no-primary control to identify detection artefact (standard IHC practice). Treat a compartment seen only at damaged edges as suspect, while retaining reproducible mixed nuclear and cytoplasmic staining for review (HPA tissue IHC; standard IHC practice).
Can this IHC stain distinguish UBA3 isoforms or an epitope affected by processing?
Do not assign staining to isoform 1 or 2 without an epitope map and isoform-specific validation; two isoforms are listed, but their antibody recognition is not established here (UniProt Q8TBC4; caption PB9838). The annotated protein chain spans residues 2–463, with N-acetylalanine at residue 2 (UniProt Q8TBC4). Those annotations do not show whether processing or acetylation changes access to the antibody’s epitope in paraffin sections (UniProt Q8TBC4; caption PB9838). If a staining pattern raises that question, compare an independently validated antibody against a distinct mapped epitope on adjacent sections (standard IHC practice). Report the observed cell and compartment pattern without attributing differences to a particular isoform or modification (standard IHC practice).
How can I check UBA3 localisation by IF alongside the chromogenic IHC result?
For a separate IF experiment, multiplex UBA3 with a validated marker of pachytene spermatocytes when examining testis, where that cell population shows high tissue IHC staining (HPA tissue IHC). Choose spectrally separated fluorophores and favor a longer-wavelength UBA3 channel if tissue autofluorescence obscures shorter-wavelength signal; include single-label and no-primary controls (standard IF practice). Because reported locations include cytosol and nucleoplasm, compare mild permeabilisation with an unpermeabilised condition if antibody access permits (HPA subcellular; standard IF practice). The side of any plasma membrane-associated epitope is not supplied, so do not interpret a negative unpermeabilised stain as absence of UBA3 (HPA subcellular; caption PB9838). Validate IF conditions independently of the paraffin-section IHC caption (caption PB9838).
How do I separate diffuse UBA3 signal from chromogenic background?
Run a no-primary section and inspect background before scoring, particularly where DAB appears outside intact cells (standard IHC practice). The illustrated PB9838 workflow used a biotinylated secondary, streptavidin-biotin complex and DAB, so check secondary-only signal and consider a biotin-independent detection method if background persists (caption PB9838; standard IHC practice). Include a peroxidase block and verify its effect with the no-primary control; that block is a general chromogenic IHC step, not UBA3-specific evidence (standard IHC practice). Titrate the primary around the illustrated 1 μg/ml condition while keeping retrieval and development time matched (caption PB9838; standard IHC practice). Interpret residual diffuse staining against the reported broad tissue expression, rather than calling every stained cell background (HPA tissue IHC).
What should I measure when UBA3 stains both nuclei and cytoplasm? ⚠ ANSWER MARKED FOR VERIFICATION
Predefine cell types and score nuclear and cytoplasmic DAB separately, since the tissue IHC profile reports staining in both compartments (HPA tissue IHC). For each compartment, record percent-positive cells and intensity categories, or calculate an H-score from percentages at intensities 0–3, yielding a 0–300 range (standard IHC practice). Normalize counts to viable cells of the same annotated type, or report positive-cell density per mm² of viable tissue when cell boundaries cannot be counted reliably (standard IHC practice). Keep exposure, DAB development and thresholds consistent across sections, and exclude folds, edges and necrotic areas before scoring (standard IHC practice). Report testis pachytene spermatocytes separately from other cells because their staining is high in the tissue profile (HPA tissue IHC).
What makes a UBA3-positive cell convincing rather than an artefact?
A convincing result follows intact cell boundaries, reproduces across sections and exceeds the no-primary control in the scored compartment (standard IHC practice). In the reported human tissue profile, pachytene spermatocytes stain highly, whereas ovarian stromal cells are not detected; use these as context rather than assuming identical patterns in every specimen (HPA tissue IHC). Nuclear and cytoplasmic signal can fit the tissue profile, and cell imaging additionally reports cytosol, plasma membrane, nucleoplasm and centrosome locations (HPA tissue IHC; HPA subcellular). Discount staining concentrated at section edges, necrotic areas or regions that remain positive without primary antibody (standard IHC practice). Check endogenous peroxidase and biotin-related background before assigning DAB signal to UBA3 in the illustrated detection workflow (caption PB9838; standard IHC practice).
Boster reagents
Best UBA3 / NEDD8-activating enzyme E1 catalytic subunit IHC Antibodies
PB9838 has IHC images from mouse and rat paraffin-embedded testis sections and an IF/ICC image from A431 cells (catalog image captions); the catalog lists human, mouse, and rat reactivity (catalog).
Real IHC data
Anti-UBE1C/UBA3 Antibody ®
Cat # PB9838
PB9838 shows IHC staining in mouse and rat paraffin-embedded testis sections (PB9838 IHC captions). PB9838 also shows IF/ICC staining in A431 cells (PB9838 IF caption).
Which to pick: Choose PB9838 for paraffin-section IHC: its images document mouse and rat testis staining, with a starting concentration of 0.5–1 μg/ml (PB9838 IHC captions; catalog dilution). The same SKU is listed for IF/ICC at 2 μg/ml, with an A431-cell IF image; its listed reactivity is human, mouse, and rat (catalog applications and reactivity; PB9838 IF caption). The IHC captions do not report the fixative, so the images establish paraffin-section use without establishing fixation (PB9838 IHC captions). The selected PB9838 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image PB9838).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8TBC4 (UBA3_HUMAN, NEDD8-activating enzyme E1 catalytic subunit).
- Human Protein Atlas. UBA3 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. UBA3 subcellular location (ICC-IF): Mainly localized to the plasma membrane and cytosol. In addition localized to the nucleoplasm and centrosome..
- Human Protein Atlas. UBA3 antibody validation summary (3 antibodies).
- The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice. The Journal of cell biology 2001 — PMC2198877.
- Inhibition of NAE-dependent protein hyper-NEDDylation in cystic cholangiocytes halts cystogenesis in experimental models of polycystic liver disease. United European gastroenterology journal 2021 — PMC8435261.
- UBA3 promotes the occurrence and metastasis of intrahepatic cholangiocarcinoma through MAPK signaling pathway. Acta biochimica et biophysica Sinica 2024 — PMC10984854.
- Comorbidity of anxiety and dry eye disease: shared biomarkers and immune responses. Frontiers in immunology 2026 — PMC13572286.
- PubMed PMID:11230166 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16641997 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for UBA3 (NEDD8-activating enzyme E1 catalytic subunit). Expected localisation Nuclear and cytoplasmic staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9838). Validated IHC antibody: PB9838 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Pachytene spermatocytes); negative Ovary. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse testis tissue; fixative not specified (datasheet PB9838); 2. Fixation: Image fixative and duration unreported (datasheet PB9838); 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 PB9838); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9838); 8. Primary antibody: Rabbit anti-UBA3, 0.5-1μg/ml (datasheet PB9838); 9. Primary incubation: Overnight at 4 °C (datasheet PB9838). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9838); Caveat: Ovarian stroma is unstained despite broad tissue expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; check epitope coverage before comparing staining (UniProt); Regulation: Ubiquitously expressed (UniProt). Evidence: UniProt Q8TBC4, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the plasma membrane and cytosol. In addition…, Datasheet PB9838, PMC10984854. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/UBA3/immunohistochemistry-ihc-guide.