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UBE3A / Ubiquitin-protein ligase E3A · IHC design guide
Design Immunohistochemistry for UBE3A
Plan UBE3A staining in paraffin sections around mainly cytoplasmic and some nuclear signal (HPA tissue IHC). This guide covers fixation consistency, the catalog antibody’s IHC conditions, and interpretation alongside HPA’s moderate staining–RNA agreement (datasheet A00582; HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
UBE3A 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 | Mainly cytoplasmic, also nuclear (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic and nuclear staining across tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A00582) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining–RNA agreement is moderate (HPA tissue IHC) | |
| Regulation | Low tissue specificity by RNA (HPA tissue RNA) | |
| Isoform / epitope | Three isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended UBE3A IHC & IF Protocols
The catalog antibody has a paraffin-section IHC protocol (datasheet A00582). Published UBE3A protocols cover immunoperoxidase staining of free-floating sections (PMC11169893) and immunogold electron microscopy (PMC6194692).
| Sample | Paraffin-embedded human gastric cancer tissue; fixative not specified (datasheet A00582) |
| Fixation | Image fixative and duration unreported (datasheet A00582); 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 A00582); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00582) |
| Primary antibody | Rabbit anti-UBE3A, 0.5-1μg/ml (datasheet A00582) |
| Primary incubation | Overnight at 4 °C (datasheet A00582) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A00582) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | UBE3A-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Mainly cytoplasmic but also nuclear expression in all tissues. No signal in the no-primary control. |
💡Decision noteFor paraffin sections, start with heat-mediated EDTA retrieval at pH 8.0 (datasheet A00582). Neither published excerpt specifies paraffin-section retrieval (PMC11169893; PMC6194692).
Section 2
What Is the Expected UBE3A Staining Pattern?
UBE3A is found in the cytoplasm and nucleus (UniProt Q05086; HPA tissue IHC). In paraffin sections, expect mainly cytoplasmic staining with some nuclear staining, including strong signal in specified glandular, respiratory epithelial and neural cell populations (HPA tissue IHC). HPA rates its tissue staining profile Approved, with medium consistency against RNA data and external verification pending (HPA tissue IHC). UBE3A has no transmembrane segment (UniProt Q05086 topology).
| Strong cytoplasmic staining with some nuclear staining in adrenal glandular cells or caudate neuronal cells. | This matches HPA's compartment pattern and its High ratings for those cell populations (HPA tissue IHC). Score the relevant cells and compartments rather than treating every cell in the section as an equally strong positive (HPA tissue IHC). |
| A crisp membrane-only rim, with little cytoplasmic or nuclear signal. | This conflicts with the cytoplasmic and nuclear locations reported for UBE3A (UniProt Q05086; HPA tissue IHC). Its lack of a transmembrane segment also makes a membrane-only interpretation unconvincing (UniProt Q05086 topology). Review morphology and controls before assigning the rim to UBE3A (general IHC practice). |
| Strong staining in adipocytes while expected positive cells stain weakly or inconsistently. | Adipocytes are Not detected in the supplied tissue profile, whereas several glandular and neural populations are High (HPA tissue IHC). Consider cross-reactivity or endogenous detection activity; inspect a no-primary control and compare cell-specific staining before scoring (general IHC practice). |
| Uniform color over cells, stroma and slide background, without a clear intracellular pattern. | This does not resolve the mainly cytoplasmic, partly nuclear pattern reported in tissue (HPA tissue IHC). Nonspecific antibody binding, inadequate washing or detection background are possible general IHC causes; the image alone cannot distinguish them (general IHC practice). |
| No signal in an HPA High population, such as bronchial respiratory epithelial cells. | A negative result there is unexpected relative to the supplied tissue profile (HPA tissue IHC). Check tissue identity, antibody performance and detection controls before calling UBE3A absent; HPA's Approved profile still has only medium RNA agreement and awaits external verification (HPA tissue IHC). |
💡Expected UBE3A appearanceCall a result positive when specified HPA High cells show strong, mainly cytoplasmic staining with some nuclear staining; a membrane-only rim or similarly strong adipocyte signal warrants scrutiny (HPA tissue IHC; UniProt Q05086 topology).
| Which pattern applies to IF/ICC? | HPA's cell-image summary places UBE3A mainly in the nucleoplasm, with additional cytosolic signal (HPA subcellular ICC-IF). That differs in emphasis from the mainly cytoplasmic tissue IHC profile (HPA tissue IHC); assess each application against its own evidence. |
| Choice of tissue and cell comparator | Adrenal, appendix, breast and cervical glandular cells are High; adipocytes are Not detected, and marrow hematopoietic cells are Low (HPA tissue IHC). Use the named cell populations when interpreting a section, since the profile does not assign one intensity to every cell in a tissue. |
| Strength of antibody evidence | HPA039410, HPA040380 and CAB009723 are each rated Approved for IHC; HPA039410 is also Approved for ICC (HPA antibodies). These ratings support use as comparators, while the tissue profile's medium RNA agreement and pending external verification limit certainty (HPA tissue IHC). |
| Protein structure and epitope interpretation | UBE3A has no signal peptide, propeptide or annotated processing beyond its full chain; it has 3 isoforms and a HECT domain at residues 776–875 (UniProt Q05086). Without an antibody epitope in this record, neither isoform coverage nor compartment-specific epitope behavior can be predicted. |
| Target-specific fixation sensitivity | No fixation-effect evidence is supplied by UniProt or HPA for this section. Antigen retrieval may be optimized as general paraffin IHC practice, but a change in UBE3A staining after retrieval would need experimental controls to establish its cause (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in an HPA High cell population. | The run may have a tissue, retrieval, antibody or detection problem; absence of UBE3A cannot be inferred from this slide alone (HPA tissue IHC; general IHC practice). | Confirm the cell population and run a known-positive section alongside a detection control; then optimize retrieval and antibody conditions within the assay (general IHC practice). |
| Only nuclear staining is visible in tissue IHC. | Nuclear UBE3A is plausible, but tissue IHC is described as mainly cytoplasmic (UniProt Q05086; HPA tissue IHC). Weak cytoplasmic signal or excess nuclear background remain possibilities (general IHC practice). | Compare with an HPA High tissue population and a no-primary control; assess nuclear and cytoplasmic signal separately (HPA tissue IHC; general IHC practice). |
| Only cytoplasmic staining is visible in tissue IHC. | The dominant compartment agrees with HPA, but its tissue profile also reports nuclear expression (HPA tissue IHC). Nuclear signal may be below this assay's detection threshold (general IHC practice). | Verify nuclear visibility against the counterstain and compare a positive control under the same staining conditions before reporting nuclear absence (general IHC practice). |
| Adipocytes stain strongly. | This disagrees with the Not detected adipocyte entry (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible, especially if the signal lacks a defined intracellular pattern (general IHC practice). | Inspect a no-primary control; if using enzyme-based detection, check the relevant endogenous-activity block and compare staining with an HPA High population (general IHC practice; HPA tissue IHC). |
| Color is diffuse across tissue and empty areas. | A field-wide deposit does not establish the expected cellular localization (HPA tissue IHC). Background from blocking, washing or the detection system is possible (general IHC practice). | Compare no-primary and reagent controls, then adjust blocking, washing or detection conditions as indicated by those controls (general IHC practice). |
| A sharply outlined cell membrane is the dominant signal. | Membrane-only staining conflicts with the reported cytoplasmic and nuclear locations and the absence of a transmembrane segment (HPA tissue IHC; UniProt Q05086 topology). | Check the no-primary control and tissue morphology, then confirm whether an independent IHC-approved antibody reproduces the cellular pattern (general IHC practice; HPA antibodies). |
Experiment evidence
IHC & IF images showing UBE3A staining
A00582 · IHCIHC analysis of UBE3A using anti-UBE3A antibody (A00582). UBE3A was detected in paraffin-embedded section of human gastric cancer tissue. He
A00582 · IHCIHC analysis of UBE3A using anti-UBE3A antibody (A00582). UBE3A was detected in paraffin-embedded section of human rectal cancer tissue. Hea
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000114062-UBE3A/tissue/cerebral+cortex#imid_9976134
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000114062-UBE3A/tissue/colon#imid_9976041
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000114062-UBE3A/tissue/liver#imid_9976050
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000114062-UBE3A/tissue/kidney#imid_9976059
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA039410; green = UBE3A, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA039410; green = UBE3A, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for UBE3A IHC & IF
🧪Run adrenal gland first: glandular cells should stain (HPA: High in adrenal gland glandular cells). Use adipose tissue as the negative comparator; adipocytes should be unstained (HPA: Not detected in adipocytes). On the positive slide, use unstained cells, if present, to assess local background rather than assuming a particular cell type is UBE3A-negative.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show UBE3A in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and nonimmune IgG controls matched to the primary antibody’s host species, clonality, and concentration; use UBE3A knockout material as a biological negative. For chromogenic detection, quench endogenous peroxidase and check for endogenous biotin background if using the caption’s avidin–biotin method (selected-SKU IHC caption: biotinylated secondary and SABC/DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state a fixative. Heat retrieval in EDTA at pH 8.0 was used for the caption’s gastric cancer section, but retrieval dependence is unreported (selected-SKU IHC caption). Frozen sections have no reported advantage here; IF offers a localisation readout, with nucleoplasm as the main location and cytosol as an additional location (HPA: approved ICC-IF locations). In adrenal sections, check for endogenous biotin signal if using avidin–biotin detection (selected-SKU IHC caption: SABC/DAB).
HPA tissue IHC evidence for UBE3A
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced UBE3A IHC Tips
Troubleshoot UBE3A staining in paraffin sections by checking retrieval, compartment pattern, background and scoring against documented IHC evidence.
What retrieval should I try first when UBE3A staining is weak?
Start with heat-mediated retrieval in EDTA pH 8.0 for paraffin sections (datasheet A00582). The documented gastric cancer tissue image used that buffer before incubation with 1 μg/ml antibody overnight at 4°C, so keep those steps consistent while assessing retrieval (datasheet A00582). Compare a retrieved section with a section that skips retrieval, using the same detection and exposure to chromogen (standard IHC practice). If staining remains weak, adjust heating and cooling cautiously on matched sections, checking tissue integrity after each change (standard IHC practice). Record cytoplasmic and nuclear signal separately because both compartments are plausible for UBE3A (UniProt Q05086 localisation; HPA tissue IHC).
Could fixation explain weak or uneven UBE3A staining?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (datasheet A00582). Record the fixative, fixation duration and processing history for each block before attributing differences to UBE3A abundance (standard IHC practice). Compare sections from blocks with matched handling, then apply the documented EDTA pH 8.0 heat retrieval consistently (datasheet A00582; standard IHC practice). Check morphology alongside stain intensity, since damaged or unevenly processed areas can complicate IHC interpretation (standard IHC practice). Do not infer a UBE3A-specific fixation effect from its cytoplasmic and nuclear distribution or its phosphorylation sites (UniProt Q05086 localisation and modified residues).
Should UBE3A staining appear in nuclei, cytoplasm, or both?
Evaluate both cytoplasmic and nuclear staining in tissue sections: the reported tissue profile is mainly cytoplasmic with nuclear expression (HPA tissue IHC). UBE3A is also annotated in the cytoplasm and nucleus, with no transmembrane segment (UniProt Q05086 localisation and topology). The cell-imaging profile emphasizes nucleoplasm with additional cytosol, so a difference in compartment emphasis across methods is possible (HPA subcellular; HPA tissue IHC). Score each compartment separately in intact cells and compare neighbouring regions processed in the same run (standard IHC practice). A signal confined to section edges or spaces without identifiable cells needs a background check before it is called UBE3A (standard IHC practice).
How can the antibody epitope affect interpretation across UBE3A isoforms?
UBE3A has 3 listed isoforms, I, II and III, but the supplied caption does not identify which each antibody detects (UniProt Q05086 isoforms; datasheet A00582). Check the antibody's stated immunogen or epitope before claiming that staining represents every isoform (standard IHC practice). The HECT domain spans residues 776–875; epitope position matters when evaluating an antibody against a protein with alternative isoforms (UniProt Q05086 domains and isoforms; standard IHC practice). Phosphoserine at 218 and ABL1-associated phosphotyrosine at 659 are annotated, but their effects on this antibody's staining are unknown (UniProt Q05086 modified residues; datasheet A00582). Report the detected UBE3A signal without assigning an isoform or modification unless the reagent has supporting evidence (standard IHC practice).
How should I plan UBE3A immunofluorescence alongside a cell-type marker?
Treat IF/ICC as a separate assay: tissue IHC establishes neither an IF dilution nor an IF fixation condition for this antibody (datasheet A00582). Multiplex UBE3A with a validated marker for the cell type under study, and verify that the primary antibodies and secondary reagents can be distinguished (standard IF practice). Choose fluorophores after inspecting unstained tissue autofluorescence, favouring a channel with lower background for the weaker signal (standard IF practice). UBE3A is annotated in nucleus and cytoplasm without a transmembrane segment, so test permeabilisation that permits access to intracellular epitopes while preserving morphology (UniProt Q05086 localisation and topology; standard IF practice). Assess nuclear and cytosolic signals separately; cell imaging reports mainly nucleoplasmic localisation with additional cytosol (HPA subcellular).
What should I check when UBE3A DAB staining is diffuse?
First compare a section processed without primary antibody with the stained section to locate detection-system background (standard IHC practice). For the documented workflow, review the 10% goat-serum block, 1 μg/ml primary incubation overnight at 4°C, and biotinylated secondary incubation for 30 minutes at 37°C (datasheet A00582). Include a peroxidase block and check for endogenous biotin-related signal when using the documented streptavidin–biotin DAB system (datasheet A00582; standard IHC practice). Adjust blocking, washes or antibody concentration on matched sections if background persists (standard IHC practice). Interpret widespread cell-associated signal cautiously because tissue IHC reports expression across tissues and low RNA tissue specificity (HPA tissue IHC).
How should I quantify UBE3A across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, then apply the same criteria to every section (standard IHC practice). Record nuclear and cytoplasmic staining separately because both are reported for UBE3A in tissue (HPA tissue IHC). For each compartment, use percent positive cells and intensity grades 0–3, or calculate an H-score on a 0–300 scale (standard IHC practice). Normalise counts to the number of evaluable cells, or report positive-cell density per mm² of evaluable tissue, excluding folds and necrosis (standard IHC practice). Keep retrieval, antibody incubation and DAB development consistent across the compared sections (datasheet A00582; standard IHC practice).
How can I distinguish genuine UBE3A staining from artefact?
Look for cell-associated cytoplasmic and nuclear staining with preserved morphology; both compartments fit the reported tissue profile (HPA tissue IHC; standard IHC practice). Compare positive regions with a no-primary control and inspect section edges, folds and necrosis before calling focal DAB deposits positive (standard IHC practice). Consider the cell type: high staining is reported in caudate neuronal cells and cerebellar granular-layer cells, while adipocytes are reported as not detected (HPA tissue IHC). Check residual endogenous peroxidase and detection-system signal when DAB persists outside convincing cellular compartments (standard IHC practice). Treat a surprising pattern as provisional because the tissue IHC reliability is Approved with medium staining–RNA consistency and pending external verification (HPA tissue IHC).
Boster reagents
Best UBE3A / Ubiquitin-protein ligase E3A IHC Antibodies
The catalog antibody has real IHC data from human paraffin sections of gastric cancer, rectal cancer, and brain tissue (A00582 IHC image captions); no IF data are supplied (A00582 catalog).
Real IHC data
Anti-UBE3A ® Antibody
Cat # A00582
A00582 is the only SKU shown and lists human reactivity and IHC among its applications (A00582 catalog). Its images show staining in paraffin sections of human gastric cancer, rectal cancer, and brain tissue (A00582 IHC image captions).
Which to pick: Choose A00582 for human paraffin-section IHC; it is a rabbit antibody with a listed IHC concentration of 0.5–1 μg/ml (A00582 catalog). The image captions describe heat retrieval in EDTA at pH 8.0, but do not report the fixative (A00582 IHC image captions). No SKU in the payload has IF/ICC validation or listed cross-species reactivity, so there is no supported IF/ICC or cross-species pick here (A00582 catalog).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q05086 (UBE3A_HUMAN, Ubiquitin-protein ligase E3A).
- Human Protein Atlas. UBE3A tissue IHC expression (reliability: Approved).
- Human Protein Atlas. UBE3A subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. UBE3A antibody validation summary (3 antibodies).
- Delayed loss of UBE3A reduces the expression of Angelman syndrome-associated phenotypes. Molecular autism 2019 — PMC6532248.
- Enhanced Transmission at the Calyx of Held Synapse in a Mouse Model for Angelman Syndrome. Frontiers in cellular neuroscience 2017 — PMC5758499.
- Regional and cellular organization of the autism-associated protein UBE3A/E6AP and its antisense transcript in the brain of the developing rhesus monkey. Frontiers in neuroanatomy 2024 — PMC11169893.
- Subcellular organization of UBE3A in human cerebral cortex. Molecular autism 2018 — PMC6194692.
- PubMed PMID:9143503 — UniProt-cited evidence.
- PubMed PMID:8988171 — UniProt-cited evidence.
- PubMed PMID:8988172 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for UBE3A (Ubiquitin-protein ligase E3A). Expected localisation Mainly cytoplasmic, also nuclear (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A00582). Validated IHC antibody: A00582 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human gastric cancer tissue; fixative not specified (datasheet A00582); 2. Fixation: Image fixative and duration unreported (datasheet A00582); 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 A00582); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00582); 8. Primary antibody: Rabbit anti-UBE3A, 0.5-1μg/ml (datasheet A00582); 9. Primary incubation: Overnight at 4 °C (datasheet A00582). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining–RNA agreement is moderate (HPA tissue IHC); Isoform / epitope: Three isoforms; epitope coverage is unspecified (UniProt); Regulation: Low tissue specificity by RNA (HPA tissue RNA). Evidence: UniProt Q05086, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A00582, PMC11169893, PMC6194692. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/UBE3A/immunohistochemistry-ihc-guide.