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RYBP / RING1 and YY1-binding protein · IHC design guide
Design Immunohistochemistry for RYBP
Plan chromogenic RYBP IHC using placental trophoblasts as a high staining reference (HPA tissue IHC). Score nuclear and cytoplasmic signal (HPA tissue IHC), and consider 5 μg/mL as the catalog antibody’s IHC starting concentration (datasheet: A04316).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RYBP Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Nuclear and cytoplasmic staining (HPA tissue IHC) | |
| Staining pattern | Placental trophoblasts show nuclear and cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Placenta+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 fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining and RNA show low consistency (HPA tissue IHC) | |
| Regulation | Reduced in breast cancer tissue (UniProt) | |
| Isoform / epitope | One full-length chain; no annotated isoforms (UniProt) |
Section 1
Recommended RYBP IHC & IF Protocols
The catalog antibody protocol is followed by published RYBP IHC methods for colorectal cancer, hepatocellular carcinoma, and mouse embryo sections (PMC10546375; PMC5244986; PMC11638158).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A04316); 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 Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear 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-RYBP, 5 μg/mL (datasheet A04316) |
| 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 | RYBP-positive staining in trophoblastic cells of placenta (HPA tissue IHC: High). HPA tissue profile: Nuclear and cytoplasmic expression in several tissues, mainly in placenta. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA at pH 9.0, 95–98 °C for 20 min (page antigen retrieval); published citrate protocols offer alternatives (PMC10546375; PMC5244986).
Section 2
What Is the Expected RYBP Staining Pattern?
RYBP is primarily nuclear, with nucleoplasmic localization and possible puncta; cytoplasmic staining is also reported (UniProt Q8N488, some puncta inferred by similarity; HPA tissue IHC; HPA ICC-IF). In paraffin sections, expect the clearest staining in placental trophoblastic cells, where HPA reports high staining (HPA tissue IHC). HPA rates its IHC evidence Approved but notes low consistency between antibody staining and RNA expression (HPA tissue IHC). RYBP has no transmembrane segment (UniProt Q8N488 topology).
| Placental trophoblastic cells show strong nuclear staining, with some cytoplasmic signal. | This fits the high trophoblast staining and nuclear plus cytoplasmic tissue profile (HPA tissue IHC). A predominantly nuclear result also fits UniProt Q8N488; compare compartments within the same cells rather than treating every cytoplasmic signal as unexpected. |
| Fine nuclear puncta appear within otherwise positive cells. | Puncta may fit the proposed Polycomb group body pattern, but that UniProt annotation is inferred by similarity (UniProt Q8N488). The supported ICC-IF location is nucleoplasm (HPA ICC-IF); puncta are therefore possible, not a required acceptance criterion for chromogenic IHC. |
| Signal is predominantly membranous or confined to extracellular material. | That distribution conflicts with the reported nuclear and cytoplasmic localization and the absence of a transmembrane segment (HPA tissue IHC; UniProt Q8N488 topology). Check section morphology and detection controls before calling it RYBP-specific staining (standard IHC practice). |
| Cells reported as unstained show signal as strong as placental trophoblasts. | For example, HPA reports adipocytes as not detected and placental trophoblasts as high (HPA tissue IHC). Unexpected staining raises possible cross-reactivity or endogenous detection activity; HPA's Approved rating and RNA–staining inconsistency warrant cautious interpretation (HPA tissue IHC; standard IHC practice). |
| Color spreads across stroma, empty spaces, and cells without a clear intracellular pattern. | Diffuse deposition lacks the reported nuclear and cytoplasmic cellular distribution (HPA tissue IHC). Consider nonspecific binding, incomplete blocking or washing, or chromogen development before scoring cells as positive (standard IHC practice). |
💡Expected RYBP appearanceCall a section positive when placental trophoblastic cells show strong, mainly nuclear staining with possible cytoplasmic signal (HPA tissue IHC; UniProt Q8N488); broad extracellular or predominantly membranous color is suspect (HPA tissue IHC; UniProt Q8N488 topology).
| Tissue and cell selection | Placental trophoblastic cells are a high-staining reference; squamous epithelial cells in esophagus, oral mucosa, tonsil and vagina are medium (HPA tissue IHC). Compare the named cell population, since a tissue-wide score can conceal differences among its cells (standard IHC practice). |
| Compartment and topology | UniProt places RYBP primarily in the nucleus and also lists cytoplasm; HPA tissue IHC reports both compartments (UniProt Q8N488; HPA tissue IHC). No transmembrane segment is annotated, so a membrane-only pattern needs control review (UniProt Q8N488 topology). |
| Strength of staining evidence | HPA calls tissue IHC Approved while reporting low consistency between antibody staining and RNA expression; the listed HPA053357 antibody is IHC Approved (HPA tissue IHC; HPA antibodies). Treat the tissue images as observed patterns, not proof that every positive cell has confirmed target-specific signal. |
| Target processing | UniProt annotates one chain spanning residues 1–228, with no signal peptide, propeptide or isoforms in this record (UniProt Q8N488). These annotations offer no basis for expecting a secreted compartment or a distinct isoform-specific staining pattern; the antibody epitope is unspecified. |
| IF/ICC Q: where should RYBP appear? | A: The supported HPA ICC-IF location is the nucleoplasm; its listed images include A-431, RT-4, U2OS and NIH 3T3 (HPA ICC-IF). IF/ICC has a separate guide; its localization evidence can inform compartment checks here without serving as an IHC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Placental trophoblasts show no convincing signal. | The expected high-staining reference is missing (HPA tissue IHC); a failed staining run or unsuitable assay conditions are possibilities (standard IHC practice). | Confirm that trophoblasts are present and intact, then review the validated IHC procedure, antibody preparation, detection reagents and a concurrent positive section (standard IHC practice). No RYBP-specific fixation sensitivity is supplied. |
| Only diffuse background appears. | Diffuse color does not match the reported cellular nuclear and cytoplasmic pattern (HPA tissue IHC); nonspecific binding or excess detection signal may contribute (standard IHC practice). | Check primary-omission and detection controls, blocking, washes and chromogen development; then reassess whether staining resolves to individual cells (standard IHC practice). |
| Signal is mainly at cell membranes. | RYBP lacks an annotated transmembrane segment and is reported in nuclear and cytoplasmic compartments (UniProt Q8N488 topology; HPA tissue IHC). | Inspect morphology and control sections, and score nuclear and cytoplasmic staining separately. Investigate persistent membrane signal as a possible assay artefact (standard IHC practice). |
| A reported unstained cell population scores strongly positive. | HPA reports adipocytes and several other specified cell populations as not detected, but its IHC evidence has low RNA–staining consistency (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible (standard IHC practice). | Verify the exact cell type and compare it with a concurrent placental section; review primary-omission and endogenous-activity controls before assigning RYBP positivity (HPA tissue IHC; standard IHC practice). |
| Placental staining is weak while surrounding background is high. | The expected high trophoblast signal has poor contrast (HPA tissue IHC). Background or insufficient effective detection may prevent a reliable cell-level call (standard IHC practice). | Review tissue preservation, assay controls, antibody dilution and detection development under the validated IHC procedure (standard IHC practice). The supplied sources give no RYBP-specific retrieval or fixation-response rule. |
| Puncta are absent despite clear nuclear staining. | Puncta are described as likely Polycomb group bodies by similarity, whereas HPA supports nucleoplasmic localization (UniProt Q8N488; HPA ICC-IF). | Assess the overall cellular and compartment pattern against the positive section; do not reject an otherwise convincing IHC result solely because discrete puncta are unresolved (HPA tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing RYBP staining
A04316 · IHCImmunohistochemistry of DEDAF in mouse liver tissue with DEDAF antibody at 5 μg/mL.
A04316 · IHCImmunohistochemistry of DEDAF in mouse liver tissue with DEDAF antibody at 10 μg/mL.
A04316 · IFImmunofluorescence of DEDAF in A549 cells with DEDAF antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000163602-RYBP/tissue/cerebral+cortex#imid_15665559
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000163602-RYBP/tissue/colon#imid_15647093
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000163602-RYBP/tissue/liver#imid_15647086
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000163602-RYBP/tissue/kidney#imid_15647087
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA053357; green = RYBP, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA053357; green = RYBP, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RYBP IHC & IF
🧪Run placenta first: trophoblastic cells should stain (HPA: High in trophoblastic cells). Use adipose tissue as the negative tissue; adipocytes should lack detectable staining (HPA: Not detected in adipocytes). On the placenta slide, assess morphologically distinct non-trophoblastic cells as internal background comparators, without assuming they are RYBP-negative.
Positive control tissue: Placenta (Trophoblastic cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RYBP in A-431, RT-4, U2OS, NIH 3T3, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a concentration-matched isotype control appropriate to the primary antibody’s host species and clonality; and, where available, a RYBP-knockout biological negative. Check endogenous peroxidase activity and, if using biotin-based detection, endogenous biotin in placenta before interpreting chromogenic signal (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or retrieval dependency is reported in the supplied evidence; optimize antigen retrieval empirically for paraffin sections. The selected A04316 tissue-IHC caption reports mouse liver staining at 5 μg/mL, but its fixative is unreported (catalog caption). Whether frozen sections or IF are easier is unreported; placental background from endogenous peroxidase or biotin may complicate chromogenic interpretation (standard IHC practice).
HPA tissue IHC evidence for RYBP
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 |
|---|---|---|---|---|
| Placenta | Trophoblastic cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Testis | Sertoli cells | Medium | Protein (IHC) | HPA → |
| Tonsil | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RYBP IHC Tips
Troubleshoot RYBP chromogenic IHC in paraffin sections by checking retrieval, compartment-specific staining, controls, and cell-level scoring before interpreting signal.
How should I retrieve RYBP in paraffin sections when nuclear staining is weak?
Use Tris-EDTA pH 9.0 heat-induced epitope retrieval at 95–98 °C for 20 min for RYBP paraffin-section IHC (page retrieval setting). Allow sections to cool consistently before blocking, and compare the same tissue across runs so retrieval changes can be distinguished from staining variation (standard IHC practice). Score nuclear signal separately from cytoplasmic signal because RYBP is primarily nuclear, although cytoplasmic localisation is also reported (UniProt Q8N488 subcellular location). If staining remains weak, trial a milder citrate buffer at pH 6.0 on paired sections as a fallback, recording whether morphology and background improve (standard IHC optimisation practice).
Could fixation explain weak or uneven RYBP staining?
RYBP-specific sensitivity to fixation is unknown from the supplied evidence; the selected tissue-IHC caption does not state its fixative (A04316 tissue-IHC caption). Record the fixative, fixation duration, specimen thickness, and time before fixation for each block, then compare sections processed under matched conditions (standard IHC practice). If nuclear staining varies across a specimen, inspect preservation and section morphology before changing antibody conditions; RYBP is primarily nuclear (UniProt Q8N488 subcellular location). Compare a known staining control from the same processing batch and change only one preparation variable at a time, since differences between batches alone cannot establish a RYBP-specific fixation effect (standard IHC practice).
Is cytoplasmic RYBP staining credible when I expect a nuclear signal?
Expect predominantly nuclear staining, potentially punctate, while recognising that cytoplasmic RYBP is also reported (UniProt Q8N488 subcellular location). The punctate nuclear pattern is proposed to represent Polycomb group bodies by similarity, so do not treat every dot as a confirmed complex (UniProt Q8N488 subcellular note). Tissue IHC reports nuclear and cytoplasmic expression in several tissues, mainly placenta, but its staining reliability is Approved with low consistency against RNA expression (HPA tissue IHC). Score nuclear and cytoplasmic compartments separately, compare them with a negative reagent control, and inspect cell borders and counterstained nuclei before interpreting diffuse cytoplasmic colour as RYBP (standard IHC practice).
Could an isoform or modified epitope explain discordant RYBP IHC?
The supplied record lists 0 annotated RYBP isoforms and a single chain spanning residues 1–228, so an isoform-specific explanation lacks support here (UniProt Q8N488 processing and isoforms). It lists phosphorylation at residues 99, 123, 127, 130, and 227, but supplies no antibody epitope mapping (UniProt Q8N488 modified residues; supplied antibody evidence). Ask for the antibody's immunogen or epitope before attributing a staining change to phosphorylation or retrieval effects (standard IHC validation practice). Compare an independent antibody recognising a documented distinct epitope, if available, and require matching cell-level localisation plus appropriate controls before treating agreement as evidence of specificity (standard IHC validation practice).
How can IF help check RYBP localisation in tissue?
Use the separate IF/ICC guide for its staining workflow, and assess whether RYBP fluorescence is concentrated in nuclei or nucleoplasm as expected (HPA subcellular: supported nucleoplasm; UniProt Q8N488 subcellular location). For multiplex tissue IF, pair RYBP with a marker identifying trophoblastic cells when examining placenta, where those cells show high tissue-IHC staining (HPA tissue IHC: High in trophoblastic cells). Choose a fluorophore channel after checking unstained-section autofluorescence, and include single-stain controls to assess bleed-through (standard IF practice). Because RYBP has no transmembrane segment and is primarily nuclear, use controlled permeabilisation to permit antibody access to the intracellular epitope, then check that nuclear morphology remains intact (UniProt Q8N488 topology and subcellular location; standard IF practice).
How do I reduce diffuse brown background without losing RYBP signal?
Start by checking whether background appears in the no-primary control; colour there points toward detection chemistry, endogenous activity, or tissue pigment rather than primary-antibody binding (standard chromogenic IHC practice). Include a peroxidase block before HRP detection, and compare DAB development times on paired sections to avoid overdevelopment (standard chromogenic IHC practice). If background occurs only with primary antibody, optimise its concentration and blocking conditions while retaining a matched positive control; the selected caption reports 5 µg/mL in mouse liver but does not report a fixative (A04316 tissue-IHC caption). Judge improvement by clearer nuclear boundaries and preserved morphology, consistent with RYBP's primarily nuclear localisation (UniProt Q8N488 subcellular location).
What is a defensible way to quantify RYBP IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define cell type and tissue region before scoring, then report the percentage of RYBP-positive nuclei and an intensity-based nuclear H-score using the same thresholds across cases (standard IHC scoring practice). Record cytoplasmic staining separately because both nuclear and cytoplasmic expression are reported, while nucleoplasm is the supported subcellular location (HPA tissue IHC; HPA subcellular). Normalise positive-cell counts to the number of evaluable cells of that type, or report positive-cell density per mm² of evaluable tissue when counts are area based (standard quantitative pathology practice). Exclude folds, necrosis, and damaged edges consistently; use batch-matched controls and identical imaging settings when comparing groups (standard quantitative IHC practice).
How can I distinguish convincing RYBP staining from artefact?
A convincing result has cell-associated signal with interpretable nuclei and an appropriate control pattern; RYBP is primarily nuclear and may appear punctate (UniProt Q8N488 subcellular location; standard IHC interpretation practice). Placental trophoblastic cells show high reported tissue-IHC staining, whereas adipocytes are listed as not detected, but neither observation alone validates an individual slide (HPA tissue IHC). Be cautious with colour restricted to cut edges, necrotic areas, or cells lacking clear boundaries, and compare any diffuse deposit with a no-primary control for endogenous enzyme signal (standard chromogenic IHC practice). Interpret unexpected cell types or wholly extranuclear patterns conservatively because HPA reports low consistency between antibody staining and RNA expression (HPA tissue IHC reliability description).
Boster reagents
Best RYBP / RING1 and YY1-binding protein IHC Antibodies
The IHC-validated antibody A04316 has mouse liver IHC images and IF images from A549 cells and mouse liver (catalog: A04316 image captions). Its listed reactivity is human, mouse and rat (catalog: A04316 reactivity).
Real IHC data
Anti-DEDAF RYBP Antibody
Cat # A04316
A04316 is the SKU with a card and its own IHC figure, showing mouse liver stained at 5 μg/mL (catalog: A04316 IHC image caption). Its IF images show A549 cells and mouse liver at 20 μg/mL (catalog: A04316 IF image captions).
Which to pick: Choose A04316 for paraffin section IHC: IHC-P is listed, and its own IHC image shows mouse liver staining at 5 μg/mL; the fixative is unreported (catalog: A04316 applications and IHC image caption). For IF/ICC work, A04316 has IF images in A549 cells and mouse liver, while M04316-3 lists ICC/IF but has no supplied IF image (catalog: A04316 IF image captions; M04316-3 applications and image captions). Both list human, mouse and rat reactivity; M04316-3 is monoclonal, and A04316 has documented mouse liver IHC and IF images (catalog: reactivity, clone and image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8N488 (RYBP_HUMAN, RING1 and YY1-binding protein).
- Human Protein Atlas. RYBP tissue IHC expression (reliability: Approved).
- Human Protein Atlas. RYBP subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. RYBP antibody validation summary (1 antibodies).
- RYBP contributes to improved prognosis in colorectal cancer via regulation of cell cycle, apoptosis and oxaliplatin sensitivity. International journal of oncology 2023 — PMC10546375.
- Expression and clinical significance of PcG-associated protein RYBP in hepatocellular carcinoma. Oncology letters 2017 — PMC5244986.
- Brachyury co-operates with polycomb protein RYBP to regulate gastrulation and axial elongation in vitro. Frontiers in cell and developmental biology 2024 — PMC11638158.
- RYBP predicts survival of patients with non-small cell lung cancer and regulates tumor cell growth and the response to chemotherapy. Cancer letters 2015 — PMC4733564.
- PubMed PMID:11395500 — UniProt-cited evidence.
- PubMed PMID:11953439 — UniProt-cited evidence.
- PubMed PMID:14765135 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RYBP (RING1 and YY1-binding protein). Expected localisation Nuclear and cytoplasmic staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A04316 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Placenta (Trophoblastic cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A04316); 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-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-RYBP, 5 μg/mL (datasheet A04316); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Antibody staining and RNA show low consistency (HPA tissue IHC); Isoform / epitope: One full-length chain; no annotated isoforms (UniProt); Regulation: Reduced in breast cancer tissue (UniProt). Evidence: UniProt Q8N488, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm., Datasheet A04316, PMC10546375, PMC5244986. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RYBP/immunohistochemistry-ihc-guide.