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CHUK / Inhibitor of nuclear factor kappa-B kinase subunit alpha · IHC design guide
Design Immunohistochemistry for CHUK
Plan CHUK chromogenic IHC in paraffin sections around cytoplasmic staining in most cell types (HPA tissue IHC). This guide covers fixation, antigen retrieval, controls and scoring, while allowing for nuclear localisation (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CHUK 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 in tissue (HPA tissue IHC); nuclear possible (UniProt) | |
| Staining pattern | Cytoplasmic staining in most cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet PB9110) | |
| Positive control | Bone marrow+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
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 and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Broad expression; induction unreported (UniProt) | |
| Isoform / epitope | One chain (1–745); no isoforms reported (UniProt) |
Section 1
Recommended CHUK IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet PB9110) with a published CHUK tissue microarray IHC protocol (PMC12647233).
| Sample | Paraffin-embedded human lung cancer tissues; fixative not specified (datasheet PB9110) |
| Fixation | Image fixative and duration unreported (datasheet PB9110); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet PB9110) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9110) |
| Primary antibody | Rabbit anti-CHUK, 0.5-1μg/ml (datasheet PB9110) |
| Primary incubation | Overnight at 4 °C (datasheet PB9110) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9110) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CHUK-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most cell types. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval for the catalog antibody (datasheet PB9110); the published protocol does not specify retrieval (PMC12647233).
Section 2
What Is the Expected CHUK Staining Pattern?
CHUK is a non-membrane protein that shuttles between cytoplasm and nucleus (UniProt O15111 topology and subcellular location). In tissue IHC, expect chiefly cytoplasmic staining across many cell types (HPA: tissue IHC profile); CHUK is widely expressed (UniProt O15111 tissue specificity). HPA rates its tissue IHC evidence Approved, with medium consistency between antibody staining and RNA expression (HPA: tissue IHC reliability).
| Cytoplasmic staining in respiratory epithelium, intestinal glands, or bone marrow hematopoietic cells. | This fits the reported tissue pattern; HPA scores these cell populations High (HPA: bronchus, small intestine, bone marrow). Compare staining within identified cells, since CHUK is broadly expressed (UniProt O15111 tissue specificity). |
| Some cells show nuclear staining alongside cytoplasmic staining. | Nuclear signal can be biologically plausible because CHUK shuttles between these compartments (UniProt O15111 subcellular location). Tissue IHC is described chiefly as cytoplasmic, while ICC-IF shows mainly nucleoplasmic signal; the two observations need not have identical emphasis (HPA: tissue IHC profile; HPA: ICC-IF summary). |
| Signal is confined to cell borders, lumens, or extracellular material, with no convincing intracellular signal. | Treat this as a suspect pattern: CHUK has no transmembrane segment or signal peptide and is assigned to cytoplasm and nucleus (UniProt O15111 topology and subcellular location). Check morphology and detection controls before scoring it as positive (standard IHC practice). |
| Strong staining appears mainly in adipocytes or fibroblasts while expected positive cells are faint. | Investigate cross-reactivity or endogenous detection activity; HPA reports Low staining in adipocytes and soft-tissue fibroblasts, but does not call them negative (HPA: tissue IHC levels). A Low reference level alone cannot establish that every stained cell is false positive. |
| Diffuse color covers tissue and empty spaces, or a known High population has no discernible signal. | Diffuse deposit cannot establish CHUK localization; assess the negative detection control and background (standard IHC practice). For absent signal, first verify the positive cells and tissue morphology against HPA's High examples, then review assay controls (HPA: tissue IHC levels; standard IHC practice). |
💡Expected CHUK appearanceCall positive when identifiable cells show mainly cytoplasmic staining, with possible nuclear signal, and clear contrast above background; High populations include bronchial respiratory epithelium and Purkinje cells (HPA: tissue IHC levels and profile; UniProt O15111 subcellular location). Isolated extracellular or border-only color is suspect (UniProt O15111 topology).
| Tissue and cell selection | Use an HPA High population, such as bone marrow hematopoietic cells or duodenal glandular cells, to judge assay performance (HPA: tissue IHC levels). Adipocytes and fibroblasts are Low references, not established negative controls (HPA: tissue IHC levels). |
| Compartment and assay context | Tissue IHC is reported as cytoplasmic in most cell types (HPA: tissue IHC profile). IF/ICC Q&A: Is nuclear CHUK plausible? Yes: nucleoplasm is its supported main ICC-IF location, with cytosol additional; CHUK shuttles between both (HPA: ICC-IF summary; UniProt O15111 subcellular location). |
| Antibody validation | Three listed antibodies have Approved IHC status; HPA001402 also has Supported ICC status (HPA: antibody validation). Approved tissue IHC still has medium staining-to-RNA consistency, so interpret an unexpected pattern with controls (HPA: tissue IHC reliability; standard IHC practice). |
| Topology and processing | CHUK has no transmembrane segment or signal peptide, and its recorded chain spans residues 1–745 (UniProt O15111 topology and processing). These annotations support intracellular interpretation; they do not specify antigen retrieval conditions or fixation sensitivity. |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in a presumed positive section. | The evaluated cells may differ from HPA's High populations, or the staining run may have failed (HPA: tissue IHC levels; standard IHC practice). | Identify the relevant cells on the section; include a documented High population and review reagent and detection controls before changing assay conditions (HPA: tissue IHC levels; standard IHC practice). |
| Color appears broadly diffuse, obscuring cell boundaries. | Background from the detection workflow can obscure intracellular staining (standard IHC practice). | Inspect a negative detection control, then review blocking, washes, primary antibody concentration, and chromogen development using the assay's established procedure (standard IHC practice). |
| Only membrane-like or extracellular color is visible. | This distribution conflicts with CHUK's annotated cytoplasmic and nuclear locations and lack of a transmembrane segment (UniProt O15111 topology and subcellular location). | Check cellular morphology and a negative detection control; do not score border-only or extracellular deposit as CHUK without convincing intracellular staining (standard IHC practice). |
| Adipocytes or fibroblasts dominate the signal. | HPA lists those populations as Low, so disproportionately strong staining warrants review; their true absence has not been established (HPA: tissue IHC levels). | Compare them with an HPA High population on the same run and examine detection controls for background or cross-reactivity (HPA: tissue IHC levels; standard IHC practice). |
| Nuclear signal is called an assay failure. | That rule overlooks CHUK nuclear shuttling and the supported nucleoplasmic ICC-IF location (UniProt O15111 subcellular location; HPA: ICC-IF summary). | Assess whether nuclear color follows cell morphology and exceeds control background; report the compartment separately from the chiefly cytoplasmic tissue IHC pattern (HPA: tissue IHC profile; standard IHC practice). |
| An unexpected pattern persists with otherwise usable controls. | Approved HPA tissue IHC evidence has medium consistency with RNA expression, so it does not resolve every discordant observation (HPA: tissue IHC reliability). | Document cell type, compartment, and control results; compare the pattern with the listed IHC-approved antibodies where available (HPA: antibody validation; standard IHC practice). |
Experiment evidence
IHC & IF images showing CHUK staining
PB9110 · IHCIHC analysis of IKKA using anti-IKKA antibody (PB9110). IKKA was detected in paraffin-embedded section of human lung cancer tissues. Heat me
M01918-4 · IHCRat kidney was stained with anti-IKK α rabbit antibody
M01918-4 · IHCHuman breast carcinoma was stained with anti-IKK α rabbit antibody
M01918-1 · IHCImmunohistochemical analysis of paraffin-embedded mouse kidney, using IKK alpha Antibody.
M01918-1 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
A01918 · IFImmunocytochemistry of IKK alpha in Jurkat cells with IKK alpha antibody at 1μg/mL.
M01918 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000213341-CHUK/tissue/cerebral+cortex#imid_529282
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000213341-CHUK/tissue/colon#imid_529192
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000213341-CHUK/tissue/liver#imid_529209
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000213341-CHUK/tissue/kidney#imid_529202
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001402; green = CHUK, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001402; green = CHUK, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CHUK IHC & IF
🧪Run bone marrow first; hematopoietic cells should stain (HPA: High in bone marrow hematopoietic cells). HPA detects CHUK in all 45 scored tissues and lists no negative tissue, so use no-primary and isotype controls; any unstained neighboring cells are a background reference, not validated CHUK-negative cells (HPA: no negative rows; standard IHC practice).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: None in HPA: CHUK is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CHUK in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Run a no-primary (secondary-only) control and a concentration-matched nonimmune rabbit IgG isotype control, alongside a matched CHUK-knockout sample if available (caption: rabbit primary and goat anti-rabbit secondary; standard IHC practice). For bone marrow DAB staining, block endogenous peroxidase and, with the caption’s avidin-biotin detection, assess endogenous biotin background (caption: SABC with DAB; standard IHC practice).
⚠️Feasibility: A CHUK-specific fixation window or fixation effect is unreported, and the selected PB9110 paraffin-section caption does not state a fixative (caption: fixative unreported). The demonstrated IHC conditions include heat retrieval in citrate buffer at pH 6 for 20 minutes; whether retrieval is essential or frozen sections are suitable is unreported (caption: citrate retrieval). HPA documents ICC-IF images and nucleoplasmic and cytosolic localization, but does not establish that IF is easier than paraffin IHC; bone marrow staining also warrants attention to endogenous peroxidase background (HPA: subcellular ICC-IF; standard IHC practice).
HPA tissue IHC evidence for CHUK
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: CHUK is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced CHUK IHC Tips
Troubleshoot CHUK staining in paraffin sections by checking retrieval, compartment, cell type, background and scoring against the available IHC evidence.
What retrieval should I use when CHUK staining is weak in paraffin sections?
Start with heat-mediated citrate retrieval at pH 6 for 20 minutes (datasheet PB9110). The selected paraffin-section example used that retrieval before overnight incubation with 1 µg/mL antibody at 4°C (datasheet PB9110). If staining remains weak, compare retrieved and unretrieved serial sections while holding antibody concentration and chromogen development constant (standard IHC practice). Include a section with an expected positive cell population, such as bronchial respiratory epithelium, and assess whether stronger signal preserves cellular detail rather than increasing diffuse background (HPA: High in bronchial respiratory epithelial cells; standard IHC practice).
Could fixation explain inconsistent CHUK staining between paraffin blocks?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not report its fixative (datasheet PB9110). Record each block’s fixative and fixation duration, then compare matched sections using the same pH 6, 20-minute citrate retrieval and antibody incubation (datasheet PB9110; standard IHC practice). For a controlled pilot, 10% neutral buffered formalin and documented fixation intervals provide a reproducible starting point, but any CHUK signal difference must be measured rather than presumed (standard IHC practice). Check morphology and staining in comparable cell populations before attributing a weak block to fixation alone (standard IHC practice).
Should I score nuclear CHUK when most tissue staining looks cytoplasmic?
Score cytoplasmic and nuclear staining separately: tissue IHC describes cytoplasmic expression in most cell types, while CHUK can shuttle between cytoplasm and nucleus (HPA tissue IHC; UniProt O15111 subcellular). Nucleoplasmic localization is also supported by cell-based IF, with cytosol as an additional location (HPA subcellular). On each section, use the counterstain to define nuclei and record the percentage of cells positive in each compartment (standard IHC practice). A nuclear result is biologically plausible, but interpret a new pattern against cell identity, morphology and controls; the tissue IHC profile alone does not establish its significance (UniProt O15111 subcellular; HPA tissue IHC; standard IHC practice).
How can epitope uncertainty affect a negative CHUK IHC result?
CHUK has 0 listed isoforms, no transmembrane segment and a kinase domain spanning residues 15–302 (UniProt O15111 isoforms, topology and domains). Its listed modified residues include 23, 176, 179 and 180, but the supplied antibody evidence does not map the recognized epitope (UniProt O15111 modified residues; datasheet PB9110). Therefore, neither a negative section nor a changed staining pattern establishes epitope masking or a particular phosphorylation state (standard IHC interpretation). Check the antibody’s documented immunogen or epitope information if available, and compare positive tissue, omitted-primary and serial-section controls before making an epitope-based explanation (standard IHC practice).
How should I adapt CHUK localisation checks to multiplex IF?
Use IF to examine cytosolic and nuclear CHUK alongside a marker that identifies the expected cell population, such as bronchial respiratory epithelial cells (UniProt O15111 subcellular; HPA: High in bronchial respiratory epithelial cells). Choose a spectrally separated fluorophore, favoring a far-red channel if the specimen shows strong shorter-wavelength autofluorescence, and check single-stain controls for bleed-through (standard IF practice). Apply and optimize permeabilisation for access to this intracellular protein; CHUK has no transmembrane segment, so a membrane-facing epitope assignment is unsupported (UniProt O15111 topology; standard IF practice). Segment nuclei with a nuclear counterstain and evaluate cytosolic signal separately, since cell-based IF supports nucleoplasm and cytosol localization (HPA subcellular; standard IF practice).
What should I check if CHUK DAB staining is diffuse or widespread?
First compare the stained section with an omitted-primary control and inspect whether DAB follows intact cells or damaged edges and debris (standard IHC practice). The selected example used 10% goat serum, a biotinylated secondary antibody, streptavidin–biotin detection and DAB (datasheet PB9110). In that workflow, check nonspecific secondary binding and endogenous biotin with appropriate controls; block endogenous peroxidase before DAB development as a general chromogenic IHC step (standard IHC practice). CHUK is widely expressed and tissue IHC reports cytoplasmic staining in most cell types, so widespread cellular signal requires compartment and control checks rather than automatic rejection (UniProt O15111 tissue specificity; HPA tissue IHC).
How should I quantify CHUK across cell types and compartments? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, then report nuclear and cytoplasmic CHUK separately (UniProt O15111 subcellular; standard IHC practice). An H-score combines the percentages of cells at intensity 0–3 to yield 0–300; alternatively, report percentage positive or positive-cell density per mm² (standard IHC practice). Normalize percentages to the number of evaluable cells of the same type, or density to the measured viable tissue area, and exclude necrotic or folded regions (standard IHC practice). Keep retrieval, imaging and thresholds consistent across sections, especially when comparing populations with different expected staining, such as high bronchial respiratory epithelium and low adipocytes (HPA tissue IHC; standard IHC practice).
How do I distinguish true CHUK staining from artefact?
Look for signal in intact cells with plausible cytoplasmic or nuclear localization; CHUK is reported in both compartments, while tissue IHC commonly shows cytoplasmic staining (UniProt O15111 subcellular; HPA tissue IHC). Compare the stained population with expected high cells, such as bronchial respiratory epithelium, and low cells, such as adipocytes, without treating either as an absolute control (HPA tissue IHC). Be cautious with staining confined to section edges, necrosis or acellular material, and investigate persistent DAB signal in an omitted-primary control for endogenous enzyme or detection background (standard IHC practice). The HPA tissue profile has medium agreement between staining and RNA expression, so a striking new compartment or cell-type pattern merits independent validation (HPA tissue IHC reliability).
Boster reagents
Best CHUK / Inhibitor of nuclear factor kappa-B kinase subunit alpha IHC Antibodies
Anti-CHUK antibodies have IHC images from human lung cancer tissue and rat and mouse kidney, plus IF/ICC images including Jurkat cells (catalog image captions).
Real IHC data
Anti-IKK alpha/CHUK Antibody ®
Cat # PB9110
Real IHC data
Anti-IKK α Rabbit Monoclonal Antibody
Cat # M01918-4
Real IHC data
Anti-IKK alpha CHUK Rabbit Monoclonal Antibody
Cat # M01918-1
Real IF data
Anti-IKK alpha CHUK Antibody
Cat # A01918
Real IF data
Anti-IKK alpha+beta CHUK Rabbit Monoclonal Antibody
Cat # M01918
PB9110 shows IHC in human lung cancer paraffin sections (PB9110 image caption); M01918-4 shows rat kidney IHC (M01918-4 image caption); M01918-1 shows mouse kidney paraffin-section IHC (M01918-1 image caption). A01918 shows Jurkat cell IF/ICC (A01918 image captions), while M01918 has IF images with no specimen identified (M01918 image captions).
Which to pick: For human paraffin-section IHC, choose PB9110: its image caption documents citrate retrieval at pH 6 for 20 minutes and DAB detection; the fixative is unreported (PB9110 image caption). For IHC across human, mouse and rat samples, consider M01918-4, which lists all three species and has a rat kidney IHC image; its caption does not report a fixative (M01918-4 catalog reactivity and image caption). For human-cell IF/ICC, A01918 has Jurkat images; M01918 is listed for IF/ICC across human, mouse and rat but targets IKK alpha+beta, and its IF captions do not identify a specimen (A01918 image captions; M01918 catalog title, applications, reactivity and image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O15111 (IKKA_HUMAN, Inhibitor of nuclear factor kappa-B kinase subunit alpha).
- Human Protein Atlas. CHUK tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CHUK subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. CHUK antibody validation summary (3 antibodies).
- Honokiol inhibits gastric cancer via tumor microenvironment modulation: a bioinformatics and single-cell analysis. Scientific reports 2025 — PMC12647233.
- Exome Sequencing of Oral Squamous Cell Carcinoma Reveals Molecular Subgroups and Novel Therapeutic Opportunities. Theranostics 2017 — PMC5399578.
- PubMed PMID:9244310 — UniProt-cited evidence.
- PubMed PMID:9252186 — UniProt-cited evidence.
- PubMed PMID:9346484 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CHUK (Inhibitor of nuclear factor kappa-B kinase subunit alpha). Expected localisation Cytoplasmic in tissue (HPA tissue IHC); nuclear possible (UniProt). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet PB9110). Validated IHC antibody: PB9110 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissues; fixative not specified (datasheet PB9110); 2. Fixation: Image fixative and duration unreported (datasheet PB9110); 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: Citrate pH 6 , 20 min (datasheet PB9110); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9110); 8. Primary antibody: Rabbit anti-CHUK, 0.5-1μg/ml (datasheet PB9110); 9. Primary incubation: Overnight at 4 °C (datasheet PB9110). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: One chain ( 1–745 ); no isoforms reported (UniProt); Regulation: Broad expression; induction unreported (UniProt). Evidence: UniProt O15111, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet PB9110, PMC12647233. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CHUK/immunohistochemistry-ihc-guide.