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RAPH1 / Ras-associated and pleckstrin homology domains-containing protein 1 · IHC design guide
Design Immunohistochemistry for RAPH1
Plan RAPH1 paraffin IHC around the cytoplasmic staining reported in most tissues (HPA tissue IHC). Compare documented positive and negative cell populations, and interpret intensity with the reported low consistency between staining and RNA expression in mind (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RAPH1 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 staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic signal across many tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A06130-1) | |
| 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 fixation consistent across sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A06130-1) | |
| Caveat | Staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | RMO1 expression is highest in liver (UniProt) | |
| Isoform / epitope | 9 isoforms; check epitope coverage across variants (UniProt) |
Section 1
Recommended RAPH1 IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 heat retrieval (datasheet A06130-1). One published RAPH1 IHC protocol provides staining conditions for wounded skin sections (PMC3542609).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A06130-1) |
| Fixation | Image fixative and duration unreported (datasheet A06130-1); 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 A06130-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A06130-1) |
| Primary antibody | Rabbit anti-RAPH1, 2-5 μg/ml (datasheet A06130-1) |
| Primary incubation | Overnight at 4 °C (datasheet A06130-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A06130-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RAPH1-positive staining in glandular cells of appendix (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A06130-1); the published method does not specify retrieval (PMC3542609).
Section 2
What Is the Expected RAPH1 Staining Pattern?
RAPH1 is a membrane-associated, cytoskeletal protein found at lamellipodia and filopodia (UniProt Q70E73; no transmembrane segment). In paraffin-section IHC, expect mainly cytoplasmic staining in many tissues (HPA tissue IHC). Examples with medium staining include appendix and breast glandular cells and cerebellar Purkinje cells (HPA tissue IHC). HPA rates its tissue IHC as Approved but reports low consistency between antibody staining and RNA expression (HPA tissue IHC).
| Cytoplasmic staining in appendix or breast glandular cells, or cerebellar Purkinje cells. | This matches cell populations scored at medium intensity by HPA (HPA tissue IHC). Compare intensity within the same cell type and staining run; a positive result need not fill every cell uniformly (general IHC practice). |
| Staining near a cell edge, alongside cytoplasmic staining. | A peripheral component is plausible: RAPH1 is recruited to membranes and localizes to lamellipodia and filopodia (UniProt Q70E73). In tissue sections, cell projections may be difficult to resolve, so edge staining alone is weak evidence of specificity (general IHC practice). |
| Strong, predominantly nuclear staining with little cytoplasmic or peripheral signal. | Question the IHC result because HPA describes tissue staining as cytoplasmic (HPA tissue IHC). Nuclear bodies appear in HPA ICC-IF data, but that location is marked uncertain (HPA subcellular). Check controls before calling a nuclear IHC pattern specific. |
| Strong staining in a cell type HPA lists as not detected, such as adipocytes. | The result conflicts with the listed tissue observation (HPA tissue IHC: adipocytes not detected). Possible explanations include cross-reactivity or endogenous detection activity (general IHC practice). Check morphology, the no-primary control, and a second antibody before assigning RAPH1. |
| Diffuse color across cells and extracellular space, or no signal in an expected-positive tissue. | Diffuse color suggests background rather than the reported cell-associated pattern (HPA tissue IHC; general IHC practice). No signal in appendix glandular cells may reflect an assay failure, but HPA's Approved rating and low RNA concordance limit how definitive one sample can be (HPA tissue IHC). |
💡Expected RAPH1 appearanceCall a section positive when defined cells show mainly cytoplasmic, potentially peripheral staining around the medium level reported for selected glandular or Purkinje cells (HPA tissue IHC; UniProt Q70E73); broad extracellular color or staining confined to unexpected cells warrants control checks (general IHC practice).
| Tissue and cell-type selection | Appendix and breast glandular cells and cerebellar Purkinje cells are medium-staining examples; adipocytes and heart cardiomyocytes are listed as not detected (HPA tissue IHC). Score named cell types, not an entire tissue as uniformly positive or negative. |
| Evidence strength | HPA labels tissue IHC Approved while reporting low consistency with RNA expression (HPA tissue IHC). Treat its staining map as a comparison point, and corroborate surprising results with controls or an independent antibody (general IHC practice). |
| Membrane association and topology | RAPH1 reaches the membrane through its PH domain and has no transmembrane segment (UniProt Q70E73). Cytoplasmic staining with a peripheral component is therefore plausible; a continuous cell-surface outline is not required to recognize the reported IHC pattern (HPA tissue IHC). |
| Isoform-dependent expression | UniProt reports nine isoforms; RMO1-RAPH1 is broadly expressed with highest levels in brain, heart, ovary and developing embryo, whereas RMO1 is highest in liver (UniProt Q70E73). Those expression summaries do not override HPA's cell-level IHC observations, including cardiomyocytes listed as not detected (HPA tissue IHC). |
| IF/ICC: What localization should I expect? | HPA reports supported plasma-membrane localization, with nuclear bodies and cytosol marked uncertain in ICC-IF (HPA subcellular). UniProt also places RAPH1 at cell projections and the cytoskeleton (UniProt Q70E73). Interpret the uncertain IF locations separately from the predominantly cytoplasmic tissue IHC pattern (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No color in appendix glandular cells. | A positive cell population is missing from this run (HPA tissue IHC: medium in appendix glandular cells); the assay may have failed (general IHC practice). | Check tissue preservation, retrieval and detection controls, then repeat with an IHC-validated antibody (general IHC practice). Do not infer target-specific fixation sensitivity from these sources. |
| Color covers the section without cell boundaries. | Diffuse chromogen can reflect nonspecific binding or detection background (general IHC practice); HPA reports a cytoplasmic cellular pattern (HPA tissue IHC). | Inspect the no-primary control and review blocking, washes and detection reagent exposure (general IHC practice). |
| Unexpected cells stain strongly while the expected cell type does not. | Cross-reactivity or endogenous detection activity is possible (general IHC practice); compare the exact cell types with HPA's tissue entries (HPA tissue IHC). | Check morphology and a no-primary control; seek agreement with an independent antibody before assigning RAPH1 (general IHC practice). |
| Only nuclei stain strongly. | Nuclear-dominant tissue IHC conflicts with HPA's cytoplasmic summary (HPA tissue IHC); nuclear bodies in ICC-IF are marked uncertain (HPA subcellular). | Review counterstain versus chromogen and compare controls and an independent antibody (general IHC practice). |
| Brown color appears in a no-primary control. | The signal can arise from endogenous enzyme activity or the detection system without primary antibody (general IHC practice). | Review the appropriate endogenous-activity block and detection reagents, then repeat the control alongside the sample (general IHC practice). |
| A heart section appears negative despite reported high expression. | UniProt reports high expression of one isoform in heart, while HPA lists cardiomyocytes as not detected by tissue IHC (UniProt Q70E73; HPA tissue IHC). | Use a documented medium-staining cell population to check assay performance before interpreting the heart result (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing RAPH1 staining
A06130-1 · IHCIHC analysis of RAPH1 using anti-RAPH1 antibody (A06130-1). RAPH1 was detected in a paraffin-embedded section of human breast cancer tissue.
A06130-1 · IHCIHC analysis of RAPH1 using anti-RAPH1 antibody (A06130-1). RAPH1 was detected in a paraffin-embedded section of human liver cancer tissue.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000173166-RAPH1/tissue/cerebral+cortex#imid_5220916
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000173166-RAPH1/tissue/colon#imid_5220834
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000173166-RAPH1/tissue/liver#imid_5220827
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000173166-RAPH1/tissue/kidney#imid_5220844
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA020027; green = RAPH1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA020027; green = RAPH1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RAPH1 IHC & IF
🧪Run breast first and expect staining in glandular cells (HPA: Medium in breast glandular cells). Use adipose tissue adipocytes as the negative comparison (HPA: Not detected in adipocytes); on the breast slide, treat non-glandular cells as internal background controls only where they remain unstained, rather than assuming every such cell is negative.
Positive control tissue: Appendix (Glandular cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RAPH1 in A-431, U-251MG, U2OS, with annotated localisation: Nuclear bodies (uncertain), Plasma membrane (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and matched rabbit IgG isotype controls alongside a RAPH1 knockout sample or a peptide-block control if the immunizing peptide is available (selected-SKU caption: rabbit primary antibody). Quench endogenous peroxidase and check the no-primary breast section for background DAB signal (selected-SKU caption: HRP/DAB detection).
⚠️Feasibility: The selected-SKU paraffin-section caption does not report a fixative or a target-specific fixation window; any fixation effect is unreported (selected-SKU caption: fixative not stated). Its demonstrated IHC conditions use heat retrieval in EDTA at pH 8.0, followed by 2 μg/ml primary antibody overnight at 4°C; whether retrieval is essential has not been established by the supplied evidence (selected-SKU caption). The supplied evidence does not establish that frozen sections or IF are easier; for breast tissue, check DAB background from endogenous peroxidase against the no-primary control (selected-SKU caption: breast tissue and HRP/DAB detection).
HPA tissue IHC evidence for RAPH1
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 |
|---|---|---|---|---|
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | Medium | Protein (IHC) | HPA → |
| Colon | Endothelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RAPH1 IHC Tips
These checks help distinguish RAPH1 staining from section preparation, detection, and interpretation artefacts in chromogenic IHC.
What retrieval should I try first when RAPH1 staining is weak in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A06130-1). The selected image used this retrieval before incubation with 2 μg/ml catalog antibody overnight at 4°C (datasheet A06130-1). If staining remains weak, compare a small retrieval time series on adjacent sections while keeping antibody concentration and detection constant (standard IHC practice). Excessive heating can damage morphology or raise nonspecific staining, so assess intact tissue and a matched negative control alongside signal intensity (standard IHC practice). The caption does not report the retrieval duration or fixative; record both for your own sections (datasheet A06130-1).
Could fixation explain inconsistent RAPH1 staining between paraffin blocks?
Target-specific fixation sensitivity for RAPH1 is unknown from the supplied evidence; the selected paraffin-section caption does not identify a fixative (datasheet A06130-1). Compare blocks with documented fixation histories using the same pH 8.0 EDTA retrieval and staining run before changing antibody concentration (datasheet A06130-1; standard IHC practice). Include an internal tissue landmark and examine nuclear detail, section integrity, and uneven staining when judging whether processing differed (standard IHC practice). Do not attribute a weak block to RAPH1 epitope loss without a controlled comparison, because HPA tissue patterns and UniProt annotations do not establish fixation sensitivity (HPA tissue IHC; UniProt Q70E73).
Which RAPH1 staining compartment should count as plausible in tissue IHC?
Expect predominantly cytoplasmic tissue staining when reading chromogenic sections, while allowing membrane-associated signal where cellular outlines are interpretable (HPA tissue IHC; UniProt Q70E73 localisation). RAPH1 is recruited to membranes through its PH domain and is associated with lamellipodia and filopodia (UniProt Q70E73 localisation). HPA reports supported plasma-membrane localisation in cell imaging, with nuclear bodies and cytosol marked uncertain; those cell-imaging observations do not establish a nuclear IHC pattern (HPA subcellular). Compare staining with adjacent cellular morphology and a counterstain before calling a thin peripheral rim specific (standard IHC practice). Score diffuse nuclear-only or extracellular pigment separately as suspect patterns rather than RAPH1-positive cells (HPA tissue IHC; standard IHC practice).
Can one IHC antibody distinguish RAPH1 isoforms or modification-dependent staining?
Do not assign a paraffin-section staining pattern to a particular RAPH1 isoform without an epitope map and isoform-specific validation (UniProt Q70E73 isoforms; standard IHC practice). The record lists 9 isoforms and places the Ras-associating and PH domains at residues 269–355 and 396–505, respectively (UniProt Q70E73). It also lists modified residues, but these annotations alone do not show that fixation, retrieval, or the catalog antibody discriminates a modification state (UniProt Q70E73; datasheet A06130-1). Compare the immunogen sequence with isoform sequences when available, then use an independent epitope or genetic control to test a proposed isoform-specific interpretation (standard IHC practice).
How should I plan a RAPH1 multiplex IF check after tissue IHC?
Treat IF as a separate validation experiment: the selected catalog evidence describes chromogenic staining of a paraffin section, with no fixative reported (datasheet A06130-1). Pair RAPH1 with a validated marker for the cell population being assessed, such as glandular or endothelial cells represented in HPA tissue observations (HPA tissue IHC; standard IF practice). Select fluorophores after checking unstained tissue for autofluorescence, and include single-channel controls to assess spectral bleed-through (standard IF practice). RAPH1 has no transmembrane segment and associates with the cytoplasmic face of membranes, so choose permeabilisation to expose the intended epitope after checking its antibody documentation (UniProt Q70E73 topology and localisation; standard IF practice).
How can I reduce diffuse brown signal without losing genuine RAPH1 staining?
Begin with the documented 10% goat-serum block and 2 μg/ml primary antibody used for the selected paraffin-section image (datasheet A06130-1). Its detection used a peroxidase-conjugated secondary and DAB; an endogenous-peroxidase block is a general chromogenic IHC control to assess, not a documented RAPH1-specific step (datasheet A06130-1; standard IHC practice). Run a no-primary section and inspect pigment, endogenous enzyme activity, and staining that follows folds or damaged edges (standard IHC practice). If background persists, adjust blocking, washing, or primary concentration one variable at a time while retaining the same retrieval and detection conditions (standard IHC practice).
What should I measure when comparing RAPH1 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because HPA describes cytoplasmic expression in most tissues and records staining in distinct glandular and endothelial populations (HPA tissue IHC). For chromogenic IHC, report the percentage of positive cells and an H-score based on intensity categories, or positive-cell density per mm² when cell counts are practical (standard IHC practice). Normalise cell counts to the number of evaluable cells in the same annotated compartment, or density to viable annotated tissue area (standard IHC practice). Keep retrieval, exposure to DAB, threshold rules, and counterstain comparable across sections; exclude folds and necrotic areas before comparing scores (standard IHC practice).
What evidence separates a convincing RAPH1-positive cell from an artefact?
A plausible positive result follows identifiable cells and fits the predominantly cytoplasmic tissue pattern, with membrane-associated staining also biologically plausible for RAPH1 (HPA tissue IHC; UniProt Q70E73 localisation). Check cell identity against the section: HPA records medium glandular staining in breast and medium endothelial staining in colon, while several other listed cell populations are not detected (HPA tissue IHC). Treat isolated nuclear-only deposits, staining restricted to tissue edges or necrosis, and signal persisting without primary antibody as possible artefacts (HPA tissue IHC; standard IHC practice). HPA rates its tissue staining Approved but reports low consistency with RNA expression; corroborate consequential calls with an independent control (HPA tissue IHC; standard IHC practice).
Boster reagents
Best RAPH1 / Ras-associated and pleckstrin homology domains-containing protein 1 IHC Antibodies
Real RAPH1 IHC images show human paraffin sections (A06130-1 image captions). The catalog lists human and mouse reactivity, but provides no IF/ICC images (A06130-1 catalog).
Real IHC data
Anti-RAPH1 Antibody ®
Cat # A06130-1
A06130-1 will render with its human breast cancer paraffin-section IHC image (A06130-1 figure caption). Its catalog also documents human liver cancer paraffin-section IHC and lists human and mouse reactivity (A06130-1 image captions; A06130-1 catalog).
Which to pick: Choose A06130-1 for human paraffin-section IHC; its image captions document EDTA retrieval at pH 8.0 and primary antibody at 2 μg/ml, while the catalog recommends 2–5 μg/ml for human IHC (A06130-1 image captions; A06130-1 catalog). The captions do not report the fixative (A06130-1 image captions). No IF/ICC option is validated here, and although A06130-1 lists mouse reactivity, its documented IHC examples are human, so mouse tissue IHC would require validation (A06130-1 applications/reactivity; A06130-1 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q70E73 (RAPH1_HUMAN, Ras-associated and pleckstrin homology domains-containing protein 1).
- Human Protein Atlas. RAPH1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. RAPH1 subcellular location (ICC-IF): Mainly localized to the nuclear bodies and plasma membrane. In addition localized to the cytosol..
- Human Protein Atlas. RAPH1 antibody validation summary (2 antibodies).
- MicroRNA-203 contributes to skin re-epithelialization. Cell death & disease 2012 — PMC3542609.
- Loss of NDR1/2 kinases impairs endomembrane trafficking and autophagy leading to neurodegeneration. Life science alliance 2023 — PMC9711861.
- PubMed PMID:11586298 — UniProt-cited evidence.
- PubMed PMID:15469845 — UniProt-cited evidence.
- PubMed PMID:15609301 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RAPH1 (Ras-associated and pleckstrin homology domains-containing protein 1). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A06130-1). Validated IHC antibody: A06130-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A06130-1); 2. Fixation: Image fixative and duration unreported (datasheet A06130-1); 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 A06130-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A06130-1); 8. Primary antibody: Rabbit anti-RAPH1, 2-5 μg/ml (datasheet A06130-1); 9. Primary incubation: Overnight at 4 °C (datasheet A06130-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A06130-1); Caveat: Staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 9 isoforms; check epitope coverage across variants (UniProt); Regulation: RMO1 expression is highest in liver (UniProt). Evidence: UniProt Q70E73, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nuclear bodies and plasma membrane. In…, Datasheet A06130-1, PMC3542609. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RAPH1/immunohistochemistry-ihc-guide.