UBE2L6 / Ubiquitin/ISG15-conjugating enzyme E2 L6 · IHC design guide

Design Immunohistochemistry for UBE2L6

Plan UBE2L6 chromogenic IHC on formalin-fixed paraffin sections with the IHC-validated antibody at 1:50–1:100 (datasheet: A07315-1). Assess cytoplasmic and nuclear staining in immune-cell subsets, using appendix lymphoid tissue or lymph-node germinal centers as positive references (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for UBE2L6 (IHC for UBE2L6): expected localisation Cytoplasmic and nuclear staining in immune-cell subsets (HPA tissue IHC), antibody A07315-1, validated IHC image, and IHC protocol steps — Printable **UBE2L6** IHC protocol sheet — expected localisation Cytoplasmic and nuclear staining in immune-cell subsets (HPA tissue IHC), antibody A07315-1, controls and protocol steps. Open the full UBE2L6 IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

UBE2L6 Immunohistochemistry Experimental Design Guide

Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.

Must know before staining

	Expected localisation	Cytoplasmic and nuclear staining in immune-cell subsets (HPA tissue IHC)
	Staining pattern	Immune-cell subsets show cytoplasmic and nuclear staining (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen)
	Positive control ⓘ	Appendix+4 more · see all
	Negative control ⓘ	Adipose tissue+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep formalin fixation consistent across paraffin sections (datasheet: A07315-1; standard IHC practice) (selected-SKU IHC image A07315-1)
	Caveat	Spleen red-pulp peroxidase may raise background (HPA tissue IHC; standard IHC practice)
	Regulation	Regulation of IHC staining intensity is unreported (UniProt)
	Isoform / epitope	2 isoforms; variant epitope coverage is unknown (UniProt)

Section 1

Recommended UBE2L6 IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet) is accompanied by 1 published UBE2L6 IHC protocol for esophageal squamous cell cancer tissue microarrays (PMC9949073).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	FFPE human hepatocarcinoma tissue (datasheet A07315-1)
Fixation	Image formalin-fixed; duration unreported (datasheet A07315-1); 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-UBE2L6, 1:50-1:100 (datasheet A07315-1)
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	UBE2L6-positive staining in lymphoid tissue of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression, most abundant in subsets of immune 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); compare pH 9.0 if staining is weak (PMC9949073).

Section 2

What Is the Expected UBE2L6 Staining Pattern?

In paraffin-section IHC, expect UBE2L6 staining in cytoplasm and nuclei, most abundant in subsets of immune cells (HPA tissue IHC: Enhanced reliability). High staining is reported in appendix lymphoid tissue, lymph-node and tonsil germinal-center cells, and splenic red-pulp cells (HPA tissue IHC). UBE2L6 has no transmembrane segment, so a membrane-only pattern would be unexpected (UniProt O14933 topology). HPA's Enhanced tissue reliability reflects agreement with RNA data, pending external verification (HPA tissue IHC).

What am I looking at on my slide?
Distinct cytoplasmic and nuclear chromogen in subsets of immune cells, especially germinal-center cells.	This fits the reported tissue pattern; lymph-node and tonsil germinal-center cells stain at a High level (HPA tissue IHC). Assess compartments within identified cells rather than treating every immune cell as an equally strong reference (HPA tissue IHC: subsets of immune cells).
An exclusively sharp plasma-membrane rim, with little cytoplasmic or nuclear staining.	This conflicts with the reported IHC compartments and the absence of a transmembrane segment (HPA tissue IHC; UniProt O14933 topology). Suspect staining artefact or nonspecific binding; morphology alone cannot identify its cause (general IHC practice).
Strong staining in adipocytes or cardiomyocytes while expected immune-cell staining is absent.	Those cell types are reported as Not detected (HPA tissue IHC). Consider cross-reactivity or endogenous detection activity, then compare with controls; unexpected staining alone cannot distinguish these explanations (general IHC practice).
Uniform pale chromogen across cells, stroma, and empty regions of the section.	A widespread haze obscures the cell-specific pattern reported by HPA (HPA tissue IHC). In chromogenic IHC, incomplete blocking, excessive antibody or detection reagent, and inadequate washing are possible causes (general IHC practice).
No detectable staining in appendix lymphoid tissue or tonsil germinal-center cells.	Both are reported High and are useful positive-reference regions (HPA tissue IHC). First check that the relevant cells are present and that the run controls worked; a blank section alone does not establish absent UBE2L6 expression (general IHC practice).

💡Expected UBE2L6 appearanceCall the IHC result consistent when cytoplasmic and nuclear staining is distinct in the expected cells, with High staining in HPA-listed immune-cell regions; a membrane-only rim or strong signal confined to HPA-listed Not detected cell types calls for review (HPA tissue IHC; UniProt O14933 topology).

How each factor affects the staining
Which tissue and cell population is scored?	HPA reports High staining in appendix lymphoid tissue, lung alveolar type I cells, lymph-node and tonsil germinal-center cells, and splenic red-pulp cells; glandular cells in adrenal gland and breast are Medium (HPA tissue IHC). Score the specified cell population, since a tissue-wide average can hide this variation (general IHC practice).
How is a low or absent comparator chosen?	Adipocytes and cardiomyocytes are Not detected, whereas thyroid and parathyroid glandular cells are Low (HPA tissue IHC). Use these categories as observed reference patterns, not as proof that every cell in a section must be unstained (HPA tissue IHC).
Which antibody supports the IHC interpretation?	CAB073407 has Enhanced IHC validation; HPA003328 has Supported IHC validation (HPA antibodies). The overall tissue profile has Enhanced reliability through agreement with RNA expression data, pending external verification (HPA tissue IHC). Record which antibody produced the result when comparing patterns.
Does IF/ICC show the same compartment?	HPA's ICC-IF summary places UBE2L6 in the cytosol, with approved cytosol localization, while tissue IHC describes cytoplasmic and nuclear expression (HPA subcellular; HPA tissue IHC). Interpret each assay against its own observation; the ICC-IF entry does not establish that every paraffin section must lack nuclear staining.
Do topology or isoforms change the expected readout?	The annotated protein lacks a signal peptide, propeptide, transmembrane segment, and glycosylation sites; UniProt lists two isoforms (UniProt O14933). These annotations provide no basis to expect a shed extracellular pattern, but they do not identify the antibody epitopes or predict isoform-specific staining (UniProt O14933).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A positive-reference immune region is blank.	The sampled section may lack the expected cell population, or an IHC step may have failed (general IHC practice); HPA reports High staining only for specified cells (HPA tissue IHC).	Confirm cell identity and section quality, review the run's positive and detection controls, then check the catalog antibody's IHC-P instructions for the actual staining conditions (general IHC practice).
Signal appears only as a membrane outline.	The pattern conflicts with cytoplasmic and nuclear tissue staining and with the lack of a transmembrane segment (HPA tissue IHC; UniProt O14933 topology). Nonspecific staining is possible (general IHC practice).	Compare serial sections and controls, inspect whether the rim tracks tissue edges, and repeat with an independently validated IHC antibody if available (general IHC practice).
Adipocytes or cardiomyocytes stain strongly.	Both are Not detected in the HPA tissue record (HPA tissue IHC). Cross-reactivity or endogenous detection activity could produce an unexpected chromogenic signal (general IHC practice).	Compare a no-primary control and an appropriate detection control; examine whether expected positive cells stain in the same run before assigning target expression (general IHC practice).
The entire section has diffuse brown background.	Excess reagent, incomplete blocking, or inadequate washing can raise nonspecific chromogen background (general IHC practice). HPA supplies no UBE2L6-specific cause for diffuse haze.	Check no-primary controls, blocking and wash steps, and antibody and detection-reagent conditions against the validated IHC-P instructions (general IHC practice).
Weak glandular staining seems inconsistent with a strong immune-cell control.	HPA reports Medium staining in adrenal and breast glandular cells and Low staining in several other glandular populations, alongside High staining in selected immune cells (HPA tissue IHC).	Score each identified cell population against its own HPA category, using the same run and counterstain to judge signal above background (HPA tissue IHC; general IHC practice).
An ICC-IF image is cytosolic, but the tissue IHC section also shows nuclear staining.	Those are the respective reported assay observations: approved cytosol localization in ICC-IF and cytoplasmic plus nuclear expression in tissue IHC (HPA subcellular; HPA tissue IHC).	Record assay and specimen type when comparing images; for this paraffin IHC readout, evaluate nuclear and cytoplasmic signal against tissue controls (HPA tissue IHC; general IHC practice).

Source: UniProt O14933 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing UBE2L6 staining

Formalin-fixed and paraffin-embedded human hepatocarcinoma reacted with UBE2L6 Antibody (N-term), which was peroxidase-conjugated to the secondary antibody, followed by DAB staining. This data demonstrates the use of this antibody for immunohistochemistry; clinical relevance has not been evaluated.

A07315-1 · IHCFormalin-fixed and paraffin-embedded human hepatocarcinoma reacted with UBE2L6 Antibody (N-term), which was peroxidase-conjugated to the sec

A07315 · IFImmunofluorescence analysis of HeLa cells using UBE2L6 antibody at dilution of 1:100. Blue: DAPI for nuclear staining.

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000156587-UBE2L6/tissue/cerebral+cortex#imid_18914901

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000156587-UBE2L6/tissue/colon#imid_18894851

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000156587-UBE2L6/tissue/liver#imid_18894844

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000156587-UBE2L6/tissue/kidney#imid_18894845

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA003328; green = UBE2L6, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for UBE2L6 IHC & IF

🧪Run tonsil first and require staining in germinal center cells (HPA: High in tonsil germinal center cells). Use heart muscle cardiomyocytes as the negative tissue (HPA: Not detected in cardiomyocytes); compare other cells on the tonsil slide with the germinal centers as internal background, without assuming those cells are UBE2L6-negative (HPA: tonsil row specifies germinal center cells only).

Positive control tissue: Appendix (Lymphoid tissue, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show UBE2L6 in A-431, U2OS, with annotated localisation: Cytosol (approved) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and a concentration-matched nonimmune control matched to the primary antibody’s host species and, for a monoclonal antibody, isotype (standard IHC practice). Confirm target specificity with a UBE2L6 knockout specimen or a validated immunizing-peptide block, and quench endogenous peroxidase before chromogenic detection in tonsil (standard IHC practice).

⚠️Feasibility: A selected antibody image explicitly reports formalin-fixed, paraffin-embedded tissue with peroxidase and DAB detection, but reports no target-specific fixation window (selected-SKU tissue-IHC caption: A07315-1). Antigen retrieval requirements and frozen-section performance are unreported in the supplied evidence; ICC-IF images in A-431 and U2OS show cytosolic localization, but do not establish that IF is easier than IHC-P (HPA: Cytosol approved; HPA: A-431 and U2OS ICC-IF images). In tonsil, endogenous peroxidase can produce chromogenic background, so interpret staining against the quenched no-primary control (standard IHC practice).

HPA tissue IHC evidence for UBE2L6

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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
Appendix	Lymphoid tissue	High	Protein (IHC)	HPA →
Lung	Alveolar cells type I	High	Protein (IHC)	HPA →
Lymph node	Germinal center cells	High	Protein (IHC)	HPA →
Spleen	Cells in red pulp	High	Protein (IHC)	HPA →
Tonsil	Germinal center cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Caudate	Glial cells	Not detected	Protein (IHC)	HPA →
Cerebellum	Cells in granular layer	Not detected	Protein (IHC)	HPA →
Cerebral cortex	Glial cells	Not detected	Protein (IHC)	HPA →
Heart muscle	Cardiomyocytes	Not detected	Protein (IHC)	HPA →

Section 3

Advanced UBE2L6 IHC Tips

Troubleshoot UBE2L6 staining in paraffin sections by checking retrieval, cell identity and compartment before comparing chromogenic signal across specimens.

What should I adjust when UBE2L6 staining is weak after antigen retrieval?

For paraffin-section IHC, start with citrate pH 6.0 heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule). Check that sections remain covered by buffer during heating, then cool them consistently and compare staining with a known positive section processed in the same run (standard IHC practice; HPA: high staining in tonsil germinal center cells). If signal remains weak, compare a second retrieval condition on adjacent sections while keeping antibody and detection conditions fixed (standard IHC practice). Excessive retrieval can damage morphology or raise background, so judge signal alongside tissue preservation and the no-primary control (standard IHC practice).

How should I troubleshoot variable UBE2L6 staining between paraffin blocks?

Target-specific fixation sensitivity for UBE2L6 is unknown; record each block’s fixative and processing history before attributing differences to antigen loss (supplied evidence: no fixation comparison; standard IHC practice). For the next collection, keep fixation and processing consistent, and stain sections from the same run together with a reference section (standard IHC practice). Compare morphology, tissue edges and internal cell populations before changing retrieval, because uneven processing can create apparent staining gradients (standard IHC practice). If archived blocks have unknown histories, report that limitation and avoid treating different staining intensities as a quantitative biological difference without matched controls (standard IHC practice).

Should I accept nuclear as well as cytoplasmic UBE2L6 staining?

Assess cytoplasmic and nuclear chromogenic signal separately: tissue IHC shows both, with expression most abundant in subsets of immune cells (HPA tissue IHC). The approved subcellular location is cytosol, so a nuclear-only pattern deserves closer scrutiny rather than automatic rejection or acceptance (HPA subcellular: cytosol; HPA tissue IHC: cytoplasmic and nuclear expression). UBE2L6 has no annotated transmembrane segment, so crisp cell-surface outlining is not an expected topology-based pattern (UniProt O14933 topology). Compare candidate cells with adjacent tissue morphology and a no-primary section, and record each compartment independently when interpreting discordant staining (standard IHC practice).

Could epitope location or splicing explain inconsistent UBE2L6 staining?

UBE2L6 has 2 annotated isoforms and a UBC core spanning residues 2–149, but the supplied evidence does not map this antibody’s precise epitope (UniProt O14933 isoforms and domains; supplied antibody evidence). The selected image describes an N-terminal antibody without specifying the recognized residues, so its staining cannot establish equal recognition of both isoforms (selected IHC caption, SKU A07315-1). Compare staining from an independently mapped epitope, if available, on adjacent sections using matched retrieval and controls (standard IHC practice). Document the antibody identity and epitope information actually available, and avoid assigning an isoform from chromogenic staining alone (standard IHC practice).

How can IF help investigate an uncertain UBE2L6 IHC pattern?

Use IF/ICC as a separate follow-up experiment, since this section’s curated workflow concerns paraffin-section chromogenic IHC (application specification). Multiplex UBE2L6 with a marker for the suspected immune population, such as CD45, and assess signal within identified cells rather than tissue-wide overlap (HPA tissue IHC: immune-cell enrichment; standard immunophenotyping practice). Choose a far-red fluorophore when tissue autofluorescence obscures shorter wavelengths, and include single-color and no-primary controls (standard IF practice). Because the approved location is cytosolic and no transmembrane segment is annotated, permeabilize fixed cells to access an intracellular epitope, then optimize conditions for that IF assay (HPA subcellular: cytosol; UniProt O14933 topology; standard IF practice).

What causes diffuse brown staining that obscures UBE2L6-positive cells?

First inspect a no-primary control and a detection-only control to separate antibody-associated signal from chromogen or detection background (standard IHC practice). Include a peroxidase-blocking step before DAB development, then compare matched sections with antibody concentration, incubation and wash conditions changed one at a time (standard chromogenic IHC practice). This matters when expected UBE2L6 signal may be cytoplasmic or nuclear and strongest in subsets of immune cells, since diffuse deposit can conceal the relevant cells (HPA tissue IHC). Treat staining at folds, damaged edges or necrotic areas cautiously, and base interpretation on intact cells with preserved morphology (standard IHC practice).

How should I score heterogeneous UBE2L6 staining in paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring, because tissue IHC reports cytoplasmic and nuclear expression enriched in subsets of immune cells (HPA tissue IHC). For a defined cell class, record percent positive cells and staining intensity, or calculate an H-score from the percentages at each intensity level (standard IHC scoring practice). For spatially dispersed positive cells, report their density per mm² of viable tissue and normalize to the area or number of eligible cells examined (standard histology practice). Apply the same threshold and imaging settings across sections, and report compartments separately so differences in cell composition do not masquerade as altered expression (standard IHC practice).

How can I distinguish convincing UBE2L6 staining from artefact?

A convincing pattern has cell-associated cytoplasmic staining, with possible nuclear staining, in morphologically intact cells; immune-cell subsets provide a relevant comparison (HPA tissue IHC; HPA subcellular: cytosol). Surface-only outlining is questionable because UBE2L6 lacks an annotated transmembrane segment, while signal restricted to the section edge or necrosis warrants a processing check (UniProt O14933 topology; standard IHC practice). Compare suspected positives with a no-primary control to assess nonspecific deposit and with a peroxidase-controlled section to assess endogenous enzyme signal (standard chromogenic IHC practice). Interpret a negative field against the cell types present: HPA reports variable tissue staining and low RNA tissue specificity, so absence in one field does not establish absence throughout the specimen (HPA tissue IHC).

Boster reagents

Best UBE2L6 / Ubiquitin/ISG15-conjugating enzyme E2 L6 IHC Antibodies

UBE2L6 antibody images cover IHC-P in formalin-fixed, paraffin-embedded human hepatocarcinoma and IF in HeLa cells (catalog image captions); IF/ICC reactivity lists human, mouse, and rat (catalog: A07315).

Real IHC data Formalin-fixed and paraffin-embedded human hepatocarcinoma reacted with UBE2L6 Antibody (N-term), which was peroxidase-conjugated to the secondary antibody, followed by DAB staining. This data demonstrates the use of this antibody for immunohistochemistry; clinical relevance has not been evaluated.

Anti-UBE2L6 Antibody (N-term)

Cat # A07315-1

View product →

Real IF data Immunofluorescence analysis of HeLa cells using UBE2L6 antibody at dilution of 1:100. Blue: DAPI for nuclear staining.

Anti-UBE2L6 Antibody

Cat # A07315

View product →

A07315-1 has an IHC-P image of formalin-fixed, paraffin-embedded human hepatocarcinoma stained with DAB (A07315-1 image caption). A07315 lists IF/ICC and human, mouse, and rat reactivity; its rendered IF image shows HeLa cells with DAPI nuclear staining (catalog: A07315 applications and reactivity; A07315 image caption).

Which to pick: Choose A07315-1 for human tissue IHC-P: it is listed as polyclonal, has a 1:50–1:100 IHC-P range, and its image documents formalin-fixed, paraffin-embedded human hepatocarcinoma (catalog: A07315-1 applications, reactivity, and dilution; A07315-1 image caption). Choose A07315 for IF/ICC, supported by its HeLa IF image and 1:50–1:200 IF range (catalog: A07315 applications and dilution; A07315 image caption). A07315 is the cross-species option because its listed reactivity includes human, mouse, and rat; the rendered image documents HeLa cells only (catalog: A07315 reactivity; A07315 image caption).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry O14933 (UB2L6_HUMAN, Ubiquitin/ISG15-conjugating enzyme E2 L6).
Human Protein Atlas. UBE2L6 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. UBE2L6 subcellular location (ICC-IF): Localized to the cytosol..
Human Protein Atlas. UBE2L6 antibody validation summary (2 antibodies).
UHRF1/UBE2L6/UBR4-mediated ubiquitination regulates EZH2 abundance and thereby melanocytic differentiation phenotypes in melanoma. Oncogene 2023 — PMC10121471.
UBE2L6 promotes invasion and metastasis of triple-negative breast cancer by enhancing autophagy through STK38 ISGylation. Cellular and molecular life sciences : CMLS 2026 — PMC13620027.
Tumor Immunophenotyping-Derived Signature Identifies Prognosis and Neoadjuvant Immunotherapeutic Responsiveness in Gastric Cancer. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 2023 — PMC10214263.
Tumor Niche Network-Defined Subtypes Predict Immunotherapy Response of Esophageal Squamous Cell Cancer. bioRxiv : the preprint server for biology 2023 — PMC9949073.
PubMed PMID:10894956 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.
PubMed PMID:16554811 — UniProt-cited evidence.