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NCBP2 / Nuclear cap-binding protein subunit 2 · IHC design guide
Design Immunohistochemistry for NCBP2
This guide outlines chromogenic IHC-P for NCBP2 using the catalog antibody at 2–5 μg/ml (datasheet A08031-2). Use its general nuclear tissue pattern and high- and low-staining cell populations to plan controls and interpret staining (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NCBP2 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 | General nuclear tissue staining (HPA tissue IHC); cytoplasm annotated (UniProt) | |
| Staining pattern | Nuclei of glandular, epithelial and hematopoietic cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A08031-2) | |
| Positive control | Adrenal gland+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 paraffin-section fixation consistent across samples (standard IHC practice; not target-specific) | |
| Caveat | Broad nuclear staining limits negative tissue controls (HPA tissue IHC) | |
| Regulation | No specific expression regulator annotated (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope coverage needs mapping (UniProt) |
Section 1
Recommended NCBP2 IHC & IF Protocols
The catalog antibody protocol is followed by two published NCBP2 IHC examples (PMC10195147; PMC11930219).
| Sample | Paraffin-embedded human bladder urothelial carcinoma tissue; fixative not specified (datasheet A08031-2) |
| Fixation | Image fixative and duration unreported (datasheet A08031-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A08031-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A08031-2) |
| Primary antibody | Rabbit anti-NCBP2, 2-5 μg/ml (datasheet A08031-2) |
| Primary incubation | Overnight at 4 °C (datasheet A08031-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A08031-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NCBP2-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet A08031-2); the published excerpts provide no retrieval buffer (PMC10195147; PMC11930219).
Section 2
What Is the Expected NCBP2 Staining Pattern?
NCBP2 is expected to show predominantly nuclear staining in paraffin sections, consistent with its nuclear cap-binding role and lack of a transmembrane segment (UniProt P52298: function, subcellular location, topology). HPA reports general nuclear expression, with high staining in several glandular, epithelial and hematopoietic cell populations (HPA: tissue IHC). Treat the pattern as supported with medium consistency between staining and RNA data (HPA: reliability).
| Clear nuclear staining in colon glandular cells or bone marrow hematopoietic cells. | This matches reported high staining in those cell populations and the general nuclear pattern (HPA: tissue IHC). Judge localization within identifiable cells, since tissue-wide color alone cannot establish a nuclear result (standard IHC practice). |
| Predominantly membrane-like or extracellular staining, with little nuclear signal. | This conflicts with the reported general nuclear IHC pattern and with a protein lacking a transmembrane segment (HPA: tissue IHC; UniProt P52298: topology). Review morphology, background and controls before calling it specific staining (standard IHC practice). |
| Strong staining appears mainly in cells expected to have low staining, while a reported high-staining population is weak. | Consider cross-reactivity or endogenous detection activity (standard IHC practice). HPA calls parathyroid glandular cells, cardiomyocytes, smooth muscle cells and adipocytes low, not absent; a single discrepant section cannot prove an off-target reaction (HPA: tissue IHC). |
| Brown signal spreads across cells and tissue spaces without clear nuclear boundaries. | Diffuse background limits interpretation of the expected nuclear pattern (HPA: tissue IHC; standard IHC practice). Check the negative control and detection steps before scoring nuclei; broad color deposition alone does not identify NCBP2-positive cells (standard IHC practice). |
| No nuclear signal appears in a section containing a reported high-staining cell population. | First check tissue identity, preservation and the assay controls (standard IHC practice). HPA reports high staining in bronchial respiratory epithelial cells and esophageal squamous epithelial cells, among others, but its supported rating is not a guarantee for every specimen (HPA: tissue IHC, reliability). |
💡Expected NCBP2 appearanceA convincing positive is clearly nuclear staining in an identifiable HPA high-staining cell population, such as colon glandular cells, with strong signal relative to background; diffuse extracellular or dominant membrane-like color is suspect (HPA: tissue IHC; UniProt P52298: topology; standard IHC practice).
| Tissue and cell selection | HPA reports high staining in adrenal, cervical, colonic, duodenal and epididymal glandular cells, plus bone marrow hematopoietic, bronchial respiratory epithelial and esophageal squamous epithelial cells (HPA: tissue IHC). Its listed low-staining cells are comparison tissue, not established negative controls (HPA: tissue IHC). |
| Strength of IHC evidence | The tissue pattern is rated Supported, with medium consistency between antibody staining and RNA expression (HPA: tissue IHC). HPA044850 has Supported IHC status; the supplied antibody list gives no IHC status for HPA062483 (HPA: antibodies). Interpret disagreement as a reason to verify the assay, not proof of absence (standard IHC practice). |
| Isoforms and epitope coverage | UniProt lists three NCBP2 isoforms and an RNA recognition motif at residues 40–118 (UniProt P52298: isoforms, domains). No antibody epitope or isoform coverage is supplied, so this record cannot predict isoform-specific staining or an antigen-retrieval requirement. |
| Detection background | For chromogenic IHC, endogenous activity and nonspecific detection can create color unrelated to the primary antibody (standard IHC practice). Use appropriate reagent controls when unexplained staining competes with the reported nuclear pattern (HPA: tissue IHC; standard IHC practice). |
| IF/ICC Q: What localization should I expect? | A: Mainly nucleoplasmic, with additional cytosol and basal body localization (HPA: subcellular ICC-IF). HPA044850 and HPA062483 have Supported ICC status (HPA: antibodies). These ICC-IF observations inform localization; they do not establish an IHC-P protocol or require visible basal bodies in tissue sections. |
| Situation | Likely cause | Next action |
|---|---|---|
| Reported high-staining tissue has no clear nuclear signal. | The section may lack the relevant cell population, or the staining run may have failed (HPA: tissue IHC; standard IHC practice). | Confirm cell identity and inspect a known-positive section and run controls; then review the IHC-validated antibody's supplied IHC-P instructions and the performed steps (standard IHC practice). |
| Signal is mostly cytoplasmic rather than nuclear. | Some cytoplasmic localization is annotated (UniProt P52298: subcellular location; HPA: ICC-IF), but cytoplasm-dominant tissue staining departs from HPA's general nuclear IHC pattern (HPA: tissue IHC). | Compare nuclear and cytoplasmic signal in identifiable cells, assess the negative control, and avoid scoring cytoplasm alone as a definitive IHC positive (standard IHC practice). |
| Membrane-like or extracellular color dominates the section. | The compartment conflicts with the general nuclear IHC profile; NCBP2 has no annotated transmembrane segment (HPA: tissue IHC; UniProt P52298: topology). Background or cross-reactivity remains possible (standard IHC practice). | Check section morphology and primary-antibody negative controls, then reassess detection background before interpreting the color as target staining (standard IHC practice). |
| Unexpectedly strong color occurs in an HPA low-staining cell population. | Low HPA staining does not mean zero expression; cross-reactivity or endogenous detection activity is also possible (HPA: tissue IHC; standard IHC practice). | Compare with a reported high-staining cell population in the same run and use appropriate primary-antibody and detection controls to investigate the discrepancy (HPA: tissue IHC; standard IHC practice). |
| Color is diffuse across the section or obscures nuclei. | High background can prevent compartment-level interpretation (standard IHC practice). The supplied NCBP2 sources do not identify a target-specific fixation effect. | Check blocking, washing, detection and counterstain balance against the assay instructions; score only cells whose nuclear boundaries remain interpretable (standard IHC practice). |
| An IF/ICC image shows cytosolic or basal body signal that differs from the IHC slide. | HPA reports those additional ICC-IF locations while describing general nuclear tissue IHC staining (HPA: subcellular ICC-IF, tissue IHC). | Assess each application against its own reported pattern; for this paraffin IHC result, prioritize identifiable nuclear staining and the tissue IHC controls (HPA: tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing NCBP2 staining
A08031-2 · IHCIHC analysis of CBP20/NCBP2 using anti-CBP20/NCBP2 antibody (A08031-2). CBP20/NCBP2 was detected in a paraffin-embedded section of human bla
A08031-2 · IHCIHC analysis of CBP20/NCBP2 using anti-CBP20/NCBP2 antibody (A08031-2). CBP20/NCBP2 was detected in a paraffin-embedded section of human adr
A08031-2 · IFIF analysis of CBP20/NCBP2 using anti-CBP20/NCBP2 antibody (A08031-2). CBP20/NCBP2 was detected in an immunocytochemical section of A549 cel
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000114503-NCBP2/tissue/cerebral+cortex#imid_12639613
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000114503-NCBP2/tissue/colon#imid_12659739
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000114503-NCBP2/tissue/liver#imid_12659740
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000114503-NCBP2/tissue/kidney#imid_12659749
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA044850; green = NCBP2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA044850; green = NCBP2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NCBP2 IHC & IF
🧪Run colon first and assess its glandular cells for NCBP2 staining (HPA: High in colon glandular cells). There is no HPA-negative tissue because NCBP2 is detected in all 45 scored tissues (HPA: no negative rows); no-primary and isotype slides carry the negative controls, while non-glandular cells on the positive slide are background comparators, not confirmed target-negative cells.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: NCBP2 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 NCBP2 in A-431, U-251MG, U2OS, MCF-7, PC-3, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and a species-matched rabbit IgG isotype control matched to the primary antibody’s clonality; use NCBP2 knockout material, if available, as a biological specificity control (selected-SKU caption: rabbit primary and goat anti-rabbit secondary; standard IHC practice). In colon, check endogenous peroxidase and background DAB signal on the no-primary slide (HPA: colon glandular cells High; standard chromogenic IHC practice).
⚠️Feasibility: The selected-SKU paraffin-section caption reports EDTA retrieval at pH 8.0, but its fixative is unreported; no target-specific fixation window or fixation effect is reported in the supplied evidence (selected-SKU tissue-IHC caption). Retrieval was used in that example, but whether NCBP2 staining depends on it is unreported (selected-SKU tissue-IHC caption). HPA has ICC-IF images showing mainly nucleoplasmic signal, but the supplied evidence does not establish whether frozen-section staining or IF is easier than paraffin IHC; in colon, assess gland-associated background on the no-primary slide (HPA: subcellular ICC-IF; HPA: colon glandular cells High; standard IHC practice).
HPA tissue IHC evidence for NCBP2
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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: NCBP2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced NCBP2 IHC Tips
Troubleshoot NCBP2 staining in paraffin sections by checking nuclear localisation, retrieval conditions, background controls and cell-specific scoring (UniProt P52298; HPA tissue IHC).
What retrieval should I try when nuclear NCBP2 staining is weak?
Begin with heat-mediated EDTA pH 8.0 retrieval for paraffin sections (datasheet A08031-2). Keep section thickness, heating conditions and cooling consistent across test and control slides, then compare nuclear signal in glandular cells against a no-primary control (standard IHC practice; HPA: High in colon glandular cells). The pictured assay used 2 μg/ml primary antibody overnight at 4°C, so avoid changing retrieval and antibody concentration in the same comparison (caption A08031-2). If nuclear staining remains weak, test an alternative retrieval condition as a documented fallback while monitoring tissue integrity and background (standard IHC practice). Score the same cell population across conditions because NCBP2 shows general nuclear expression (HPA tissue IHC).
Could fixation explain weak NCBP2 staining in my paraffin sections?
The pictured NCBP2 section is paraffin-embedded, but its fixative is unreported; NCBP2-specific fixation sensitivity is therefore unknown (caption A08031-2). Record the fixative and fixation duration for each specimen, and compare sections processed together before attributing a staining difference to NCBP2 abundance (standard IHC practice). Inspect nuclear morphology and retrieval consistency, since damaged or unevenly processed sections make nuclear scoring unreliable (standard IHC practice; HPA tissue IHC: general nuclear expression). The EDTA pH 8.0 retrieval and 2 μg/ml primary concentration provide documented assay starting points, but do not establish an optimal fixation condition (caption A08031-2). Use matched processing controls when comparing specimens (standard IHC practice).
How should I assess staining outside the nucleus?
Prioritise nucleoplasmic staining when assessing paraffin sections because tissue IHC shows general nuclear expression and subcellular data support the nucleoplasm as the main location (HPA tissue IHC; HPA subcellular). Some cytoplasmic signal is biologically plausible because UniProt lists nucleus and cytoplasm, and HPA additionally supports cytosol localisation (UniProt P52298; HPA subcellular). Assess nuclear and cytoplasmic staining separately in intact cells, using the counterstain to identify nuclei and excluding damaged section edges (standard IHC practice). Diffuse staining across tissue spaces or staining that persists in a no-primary control needs background investigation before interpretation (standard IHC practice). A basal-body signal reported in cell imaging should not be assumed identifiable in routine chromogenic sections (HPA subcellular; standard IHC practice).
Could isoforms or epitope accessibility change the staining pattern?
NCBP2 has 3 annotated isoforms and an RNA recognition motif spanning residues 40–118 (UniProt P52298). Its recorded modifications include phosphoserines at 13 and 18 and methylarginine at 146, but the supplied antibody caption does not map its epitope (UniProt P52298; caption A08031-2). Consequently, a change in staining cannot establish isoform-specific recognition or modification-dependent masking without additional validation (standard IHC interpretation). Compare matched sections under the documented EDTA pH 8.0 retrieval and inspect whether nuclear staining remains consistent across cells (caption A08031-2; HPA tissue IHC). If patterns diverge, request epitope information and test an independently validated antibody before assigning a molecular cause (standard IHC practice).
How can IF help check the cellular source of an IHC signal?
Use IF as a separate localisation check: NCBP2 is mainly nucleoplasmic, with additional cytosolic and basal-body locations reported in cell imaging (HPA subcellular). For multiplex imaging, pair NCBP2 with a marker that identifies the expected cell population, such as glandular cells in colon, and compare signals within individual cells (HPA: High in colon glandular cells; standard IF practice). Choose a fluorophore channel after checking tissue autofluorescence and spectral overlap; a far-red channel can be useful when shorter-wavelength background is strong (standard IF practice). Permeabilisation should allow antibody access to the nucleoplasmic epitope; NCBP2 has no transmembrane segment, and the antibody epitope is unreported (UniProt P52298; caption A08031-2). Interpret IF alongside its own controls rather than treating the paraffin-section caption as IF validation (caption A08031-2; standard IF practice).
What should I check when DAB staining is diffuse or widespread?
First compare the stained section with a no-primary control and inspect whether DAB follows intact nuclei or appears across stroma, damaged edges and tissue spaces (standard IHC practice; HPA tissue IHC: general nuclear expression). The pictured assay used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and an HRP/DAB detection workflow (caption A08031-2). Check the peroxidase block and detection-only control when signal appears without primary antibody; these are general chromogenic IHC controls, not evidence of NCBP2 expression (standard IHC practice). If background rises with primary antibody, compare a lower concentration against the documented setting while preserving the same retrieval and exposure (caption A08031-2; standard IHC practice). Reassess nuclear contrast before scoring (HPA tissue IHC).
How should I quantify NCBP2 across sections with different cellularity? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, then report nuclear percentage positive, an intensity-weighted H-score, or positive-cell density per mm² (standard IHC practice; HPA tissue IHC: general nuclear expression). Normalise percentage and H-score to eligible intact cells in that population, and normalise density to the measured tissue area (standard IHC practice). Record cytoplasmic signal separately because NCBP2 also has cytoplasmic localisation evidence, while nucleoplasm is its supported main location (UniProt P52298; HPA subcellular). Apply one threshold, counterstain assessment and exclusion rule for edges or necrosis across compared sections (standard IHC practice). Include matched controls and report the scoring compartment, since mixed-cell fields can obscure cell-specific differences (standard IHC practice).
When is an apparent NCBP2-positive cell convincing?
A convincing IHC signal identifies intact nuclei in the scored cell population and exceeds the matched no-primary background (HPA tissue IHC: general nuclear expression; standard IHC practice). Strong staining solely in tissue spaces, necrotic regions or cut edges warrants artefact checks before assigning NCBP2 positivity (standard IHC practice). Cytoplasmic staining alone is inconclusive: cytoplasmic localisation is recorded, but nucleoplasm is the supported main site in cell imaging (UniProt P52298; HPA subcellular). Check a detection-only control for endogenous enzyme-related DAB signal, and compare the same tissue compartment across processing-matched sections (standard IHC practice). HPA rates tissue staining as Supported with medium staining–RNA consistency, so resolve unexpected patterns with independent evidence (HPA tissue IHC).
Boster reagents
Best NCBP2 / Nuclear cap-binding protein subunit 2 IHC Antibodies
A08031-2 has IHC images of human paraffin sections and an IF/ICC image of A549 cells (A08031-2 image captions). Its stated reactivity includes human, mouse, and rat (catalog reactivity).
Real IHC data
Anti-CBP20/NCBP2 Antibody ®
Cat # A08031-2
A08031-2 has IHC images from human bladder urothelial carcinoma and adrenocortical adenoma paraffin sections (A08031-2 IHC captions). Its IF/ICC image uses A549 cells (A08031-2 IF caption).
Which to pick: For tissue IHC, choose A08031-2: its IHC captions document EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody on human paraffin sections; the fixative is unreported (A08031-2 IHC captions). For IF/ICC, A08031-2 has an A549 cell image using 5 μg/ml primary antibody (A08031-2 IF caption). For mouse or rat studies, A08031-2 is listed as reactive, but its supplied IHC and IF images show human material (catalog reactivity; A08031-2 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P52298 (NCBP2_HUMAN, Nuclear cap-binding protein subunit 2).
- Human Protein Atlas. NCBP2 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. NCBP2 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol and basal body..
- Human Protein Atlas. NCBP2 antibody validation summary (2 antibodies).
- Expression pattern and prognostic value of key regulators for N7-methylguanosine RNA modification in prostate cancer. Acta biochimica et biophysica Sinica 2023 — PMC10195147.
- Integrated Analysis of Tumor Mutation Burden and Immune Infiltrates in Hepatocellular Carcinoma. Diagnostics (Basel, Switzerland) 2022 — PMC9406847.
- Comprehensive investigation in oncogenic functions and immunological roles of NCBP2 and its validation in prostate cancer. Translational oncology 2024 — PMC11283080.
- NCBP2 predicts the prognosis and the immunotherapy response of cancers: a pan-cancer analysis. PeerJ 2025 — PMC11930219.
- PubMed PMID:7651522 — UniProt-cited evidence.
- PubMed PMID:7478990 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NCBP2 (Nuclear cap-binding protein subunit 2). Expected localisation General nuclear tissue staining (HPA tissue IHC); cytoplasm annotated (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A08031-2). Validated IHC antibody: A08031-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular 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 bladder urothelial carcinoma tissue; fixative not specified (datasheet A08031-2); 2. Fixation: Image fixative and duration unreported (datasheet A08031-2); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A08031-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A08031-2); 8. Primary antibody: Rabbit anti-NCBP2, 2-5 μg/ml (datasheet A08031-2); 9. Primary incubation: Overnight at 4 °C (datasheet A08031-2). Watch for: Fixation: Keep paraffin-section fixation consistent across samples (standard IHC practice; not target-specific); Caveat: Broad nuclear staining limits negative tissue controls (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage needs mapping (UniProt); Regulation: No specific expression regulator annotated (UniProt). Evidence: UniProt P52298, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A08031-2, PMC10195147, PMC11930219. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NCBP2/immunohistochemistry-ihc-guide.