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RRAD / GTP-binding protein RAD · IHC design guide
Design Immunohistochemistry for RRAD
Plan RRAD staining in paraffin sections using the observed cytoplasmic pattern and its uncertain reliability (HPA tissue IHC). Start the IHC-validated antibody at 1:100–1:300 (datasheet), then compare test sections with matched controls (standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RRAD 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 | Predominantly cytoplasmic; nuclear in a few tissues (HPA tissue IHC) | |
| Staining pattern | General cytoplasmic staining, with some nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Bronchus+4 more · see all | |
| Negative control | Adipose tissue+3 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A30715) | |
| Caveat | Staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Higher in type II diabetic muscle (UniProt) | |
| Isoform / epitope | One 1–308 chain; no isoforms or transmembrane segment (UniProt) |
Section 1
Recommended RRAD IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by two published RRAD IHC protocols (PMC6923381; PMC10024404).
| Sample | Paraffin-embedded human heart tissue; fixative not specified (datasheet A30715) |
| Fixation | Image fixative and duration unreported (datasheet A30715); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-RRAD, 1:100 - 1:300 (datasheet A30715) |
| 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 | RRAD-positive staining in respiratory epithelial cells of bronchus (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression with additional nuclear expression in a few tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen retrieval; PMC6923381 reports 97 °C for 20 min).
Section 2
What Is the Expected RRAD Staining Pattern?
RRAD is annotated at the cell membrane without a transmembrane segment (UniProt P55042 topology). In tissue IHC, expect predominantly cytoplasmic staining in the cell populations HPA reports as positive, including respiratory epithelial cells and placental decidual cells (HPA: tissue IHC). Interpret the pattern cautiously: HPA rates tissue IHC reliability “Uncertain” because staining and RNA expression show low consistency (HPA: tissue IHC).
| Distinct cytoplasmic staining in bronchial respiratory epithelial cells or placental decidual cells. | These are reported High staining populations (HPA: tissue IHC). Cell-associated signal with visible unstained structures supports an interpretable IHC result; HPA’s “Uncertain” reliability still limits confidence that every stained cell contains RRAD (HPA: tissue IHC). |
| Predominantly nuclear staining, with little cytoplasmic or membrane-associated signal. | Recheck this pattern before assigning it to RRAD: HPA describes general cytoplasmic tissue staining and nuclear staining in only a few tissues (HPA: tissue IHC). Nucleoplasmic localisation is reported in ICC/IF, so a nuclear component alone does not prove artefact (HPA: subcellular ICC/IF). |
| Strong staining in a cell population reported as “Not detected,” such as adipocytes. | This conflicts with the reported adipocyte IHC result (HPA: adipose tissue IHC). Consider antibody cross-reactivity or endogenous chromogenic activity, then compare cell identity and staining against controls (general IHC practice). The HPA tissue assay is rated “Uncertain” (HPA: tissue IHC). |
| Diffuse colour across tissue and empty spaces, with poor separation between cells. | This is difficult to score as cell-specific RRAD staining. Evaluate background from detection reagents, endogenous activity, or excess antibody using appropriate control sections (general IHC practice). The reported tissue pattern is cellular and generally cytoplasmic (HPA: tissue IHC). |
| No staining in a section expected to contain bronchial respiratory epithelial cells. | HPA reports High staining in those cells, making them a useful comparator, though not an independently validated positive control (HPA: bronchus IHC; HPA: tissue IHC reliability “Uncertain”). Check tissue preservation, retrieval, antibody application, and chromogenic detection as general IHC workflow steps. |
💡Expected RRAD appearanceCall a result consistent with the reported tissue pattern when staining is cell-associated and predominantly cytoplasmic in an HPA High population such as bronchial respiratory epithelium; nuclear-only colour or diffuse colour without cell boundaries warrants review (HPA: tissue IHC; general IHC interpretation).
| Tissue and cell selection | HPA reports High staining in bronchial and nasopharyngeal respiratory epithelium, oral squamous epithelium, fallopian tube glandular cells, and placental decidual cells (HPA: tissue IHC). Specify the cell population when scoring; a whole-section score can hide that distinction (general IHC practice). |
| Compartment and topology | UniProt annotates RRAD at the cell membrane and lists no transmembrane segment (UniProt P55042 topology). HPA describes general cytoplasmic tissue staining (HPA: tissue IHC). Evaluate compartment alongside tissue morphology; a crisp membrane outline is not required by the reported IHC pattern. |
| Strength of tissue evidence | HPA assigns “Uncertain” tissue IHC reliability because antibody staining and RNA expression have low consistency (HPA: tissue IHC). Its listed antibody, HPA041755, is “Uncertain” for IHC and “Approved” for ICC (HPA: antibody validation). Treat tissue positives as reported observations, not definitive specificity proof. |
| Expression and assay disagreement | UniProt reports greatest abundance in heart and detection in adipose tissue (UniProt P55042 tissue specificity). HPA reports group-enriched heart and skeletal muscle RNA, but adipocytes as “Not detected” by tissue IHC (HPA: tissue IHC). Avoid using abundance or RNA alone to predict a slide’s staining intensity. |
| IF/ICC Q: Where should RRAD appear? | A: Mainly at the plasma membrane, with additional nucleoplasmic, Golgi, and flagellar centriole localisation (HPA: subcellular ICC/IF). HPA rates the listed antibody “Approved” for ICC, while its IHC rating is “Uncertain” (HPA: antibody validation); interpret each assay against its own reported pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive epithelium has little or no chromogenic signal. | The tissue may lack the intended cell population, or a general IHC workflow step may have failed. Bronchial respiratory epithelial cells are reported High (HPA: bronchus IHC). | Confirm the cells on the counterstain, then review section condition, retrieval, antibody incubation, and detection with a working control (general IHC practice). Do not infer RRAD-specific retrieval sensitivity from the HPA result. |
| Colour is strongest in nuclei throughout the section. | A widespread nuclear-only pattern exceeds HPA’s description of nuclear expression in a few tissues (HPA: tissue IHC). Nucleoplasm is an additional ICC/IF location (HPA: subcellular ICC/IF). | Compare nuclei and cytoplasm within identified cell populations; inspect negative controls and scoring criteria before calling the IHC pattern positive (general IHC practice). |
| Adipocytes stain strongly while the expected positive population is weak. | HPA reports adipocytes as “Not detected” by IHC, although UniProt reports RRAD detection in adipose tissue (HPA: adipose tissue IHC; UniProt P55042 tissue specificity). | Verify cell identity and compare controlled sections. Report the assay disagreement rather than treating either source as proof that the strong adipocyte stain is specific. |
| Brown colour is diffuse, including spaces without cells. | Background from antibody or detection reagents, or endogenous chromogenic activity, can obscure localisation (general IHC practice). | Inspect no-primary and detection controls, confirm blocking and washing, and score only signal tied to identifiable cells (general IHC practice). |
| A membrane outline is faint despite cytoplasmic staining in reported positive cells. | HPA’s tissue IHC profile is generally cytoplasmic, whereas its ICC/IF profile is mainly plasma membrane (HPA: tissue IHC; HPA: subcellular ICC/IF). | Score the IHC slide against the tissue pattern and cell identity. Record membrane accentuation if visible; do not require an ICC/IF-like outline to accept tissue staining. |
| Replicate sections disagree in staining intensity or location. | The HPA tissue IHC assessment itself is “Uncertain” because staining and RNA data have low consistency (HPA: tissue IHC). Routine section or detection variation may also contribute (general IHC practice). | Compare the same identified cell population across sections, include appropriate controls, and document the disagreement before drawing a biological conclusion (general IHC practice). |
Experiment evidence
IHC & IF images showing RRAD staining
A30715 · IHCImmunohistochemistry analysis of paraffin-embedded human heart tissue, using RAD Antibody. The picture on the right is blocked with the synt
A30715 · IFImmunofluorescence analysis of A549 cells, using RAD Antibody. The picture on the right is blocked with the synthesized peptide.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000166592-RRAD/tissue/cerebral+cortex#imid_11265883
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000166592-RRAD/tissue/colon#imid_11251625
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000166592-RRAD/tissue/liver#imid_11251626
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000166592-RRAD/tissue/kidney#imid_11251643
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA041755; green = RRAD, blue = nucleus, red = microtubules.
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA041755; green = RRAD, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RRAD IHC & IF
🧪Run bronchus first and score respiratory epithelial cells for RRAD staining (HPA: High in bronchus respiratory epithelial cells). Use adipose tissue adipocytes as the biological negative (HPA: Not detected in adipocytes); on the bronchus slide, compare adjacent non-epithelial cells for background staining without treating them as a validated RRAD-negative population (HPA: High is reported for respiratory epithelial cells).
Positive control tissue: Bronchus (Respiratory epithelial cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RRAD in A-549, SiHa, U2OS, hTERT-RPE1 (serum starved), Sperm, with annotated localisation: Plasma membrane (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-species- and clonality-matched isotype controls, plus a knockout sample or peptide-blocked primary antibody as a specificity control (standard IHC practice; A30715 caption: peptide-blocked heart section). Quench and check endogenous peroxidase activity in the bronchus section before interpreting chromogenic signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A30715 paraffin heart caption does not state a fixative (A30715 caption: fixative not stated). Retrieval dependence and whether frozen sections or IF are easier than paraffin IHC are unreported; HPA documents plasma-membrane ICC-IF localization without establishing comparative ease (HPA: plasma membrane approved). Endogenous peroxidase activity can confound chromogenic interpretation in bronchus, making the no-primary control useful when scoring respiratory epithelial cells (standard IHC practice; HPA: High in bronchus respiratory epithelial cells).
HPA tissue IHC evidence for RRAD
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — 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 |
|---|---|---|---|---|
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuropil | High | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | High | Protein (IHC) | HPA → |
| Nasopharynx | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Seminal vesicle | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RRAD IHC Tips
Troubleshoot RRAD staining in paraffin sections by checking retrieval, controls, cellular distribution, and scoring before interpreting chromogenic signal.
What should I change when RRAD staining is weak after retrieval?
Start with citrate pH 6.0 heat-induced retrieval at 95–98 °C for 20 min (page retrieval setting: A30715). Keep section thickness, cooling, and detection conditions constant while comparing retrieved sections with an otherwise matched control, so changes in signal can be attributed to retrieval. The catalog antibody has a paraffin-section heart image with peptide blocking, but its caption does not report retrieval conditions (A30715 tissue-IHC caption). If staining remains weak, test a longer citrate retrieval or an alternative buffer on adjacent sections, checking for tissue damage and increased background before adopting either condition.
How should I assess whether fixation is causing weak RRAD IHC?
RRAD-specific sensitivity to fixation is unknown from the supplied evidence; the paraffin-section heart caption does not identify a fixative or fixation duration (A30715 tissue-IHC caption). Record the fixative, time in fixative, and processing history for each specimen before comparing staining between cases. On adjacent sections, hold citrate pH 6.0 retrieval at 95–98 °C for 20 min and the antibody and detection conditions constant (page retrieval setting: A30715). If sections with different fixation histories stain differently, repeat with matched controls before attributing that difference to RRAD abundance or a specific fixation effect.
Should RRAD staining be membranous, cytoplasmic, or nuclear?
Assess membrane-associated staining first: UniProt places RRAD at the cell membrane without an annotated transmembrane segment, and HPA approves plasma-membrane localisation (UniProt P55042 topology and subcellular record; HPA subcellular). HPA tissue IHC also describes general cytoplasmic staining and nuclear staining in a few tissues, but rates tissue-IHC reliability as uncertain (HPA tissue IHC). Score membrane, cytoplasmic, and nuclear signal separately rather than combining them into one positive category. When a compartment dominates unexpectedly, compare peptide-blocked and unblocked sections and inspect neighboring cells, tissue edges, and counterstain before assigning that pattern to RRAD (A30715 tissue-IHC caption).
Could epitope accessibility explain inconsistent RRAD staining?
The supplied UniProt record lists one 1–308 chain and no annotated isoforms, so an isoform-specific explanation lacks support here (UniProt P55042 processing and isoforms). It reports methylation at residue 24 and phosphorylation at residue 26, but the catalog antibody's epitope is not specified in the supplied caption (UniProt P55042 modified residues; A30715 tissue-IHC caption). Do not assume that either modification changes binding without epitope mapping or a direct comparison. The heart image includes a synthesized-peptide block; reproduce that control where feasible, then compare adjacent sections under identical retrieval and detection conditions (A30715 tissue-IHC caption).
How can IF help resolve an ambiguous RRAD IHC pattern?
In a parallel IF experiment, pair RRAD with a validated marker for the expected cell type, such as a cardiomyocyte marker in heart, and inspect signal within the same cells (UniProt P55042 tissue specificity). Choose fluorophores after checking unstained tissue for autofluorescence, and include single-label controls to assess channel bleed-through. HPA reports approved plasma-membrane localisation with additional nucleoplasm, Golgi, and flagellar-centriole localisation, so compare compartment patterns cautiously (HPA subcellular). Because the antibody epitope's position relative to the membrane is not supplied, assess mild permeabilisation against an unpermeabilised condition when testing intracellular access; do not infer that requirement from RRAD's lack of a transmembrane segment (UniProt P55042 topology).
How do I separate RRAD signal from chromogenic background?
Run a no-primary control through the same chromogenic detection steps to reveal signal from the tissue or detection reagents, and use a peroxidase block when applying a peroxidase-based DAB workflow. Compare an unblocked section with peptide-blocked material where available; the selected heart image includes such a block (A30715 tissue-IHC caption). Inspect diffuse precipitate, folds, section edges, and damaged areas before counting cells, then adjust blocking, washing, or detection exposure one variable at a time. HPA rates tissue-IHC reliability uncertain because staining and RNA expression have low consistency, so a visually dark section alone is weak evidence of RRAD specificity (HPA tissue IHC).
What is a defensible way to score RRAD in IHC sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, then report the percentage of positive cells and an intensity-weighted H-score for that same population. Use identical staining, imaging, and threshold settings across sections, and normalize positive counts to the number of eligible cells or sampled tissue area in mm². Keep membrane, cytoplasmic, and nuclear scores separate because the reported distributions differ across sources (UniProt P55042 subcellular record; HPA tissue IHC; HPA subcellular). Include matched controls and record excluded folds, edges, and necrotic regions; interpret small between-group differences cautiously because HPA rates tissue-IHC reliability uncertain (HPA tissue IHC).
When does positive RRAD staining warrant a biological conclusion?
Give greatest weight to a reproducible cellular pattern that fits the sampled tissue and is reduced by peptide blocking; peptide-blocked heart tissue is shown for the catalog antibody (A30715 tissue-IHC caption). Check whether membrane-associated signal fits RRAD's annotated localisation, while allowing for reported cytoplasmic and occasional nuclear staining (UniProt P55042 subcellular record; HPA subcellular; HPA tissue IHC). Treat signal confined to an unexpected cell population, section edge, necrotic area, or no-primary control as a reason to investigate artefact, including endogenous enzyme activity. HPA reports low consistency between staining and RNA expression, so validate biological comparisons with matched sections and an independent measure where possible (HPA tissue IHC).
Boster reagents
Best RRAD / GTP-binding protein RAD IHC Antibodies
A30715 has IHC data from paraffin-embedded human heart tissue and IF data from A549 cells (catalog image captions). Its listed reactivity covers human, mouse, and rat (catalog).
Real IHC data
Anti-RAD RRAD Antibody
Cat # A30715
A30715 will render with an IHC image of paraffin-embedded human heart tissue, including a peptide-blocked comparison (catalog IHC caption). A30715 also has an IF image of A549 cells; IHC, IF, and ICC are listed applications, and human, mouse, and rat are listed species (catalog IF caption; catalog).
Which to pick: For tissue IHC, choose A30715: its own image shows paraffin-embedded human heart tissue, but the fixative is unreported (catalog IHC caption). For IF/ICC, A30715 lists both applications and shows IF in A549 cells (catalog applications; catalog IF caption). For work across species, A30715 lists human, mouse, and rat reactivity and is polyclonal; the supplied IHC image documents human tissue only (catalog reactivity; catalog dilution record; catalog IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P55042 (RAD_HUMAN, GTP-binding protein RAD).
- Human Protein Atlas. RRAD tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. RRAD subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to the nucleoplasm, the Golgi apparatus and flagellar centriole..
- Human Protein Atlas. RRAD antibody validation summary (1 antibodies).
- Ras-related associated with diabetes gene acts as a suppressor and inhibits Warburg effect in hepatocellular carcinoma. OncoTargets and therapy 2016 — PMC4935086.
- RRAD suppresses the Warburg effect by downregulating ACTG1 in hepatocellular carcinoma. OncoTargets and therapy 2019 — PMC6400130.
- RRAD expression in gastric and colorectal cancer with peritoneal carcinomatosis. Scientific reports 2019 — PMC6923381.
- SETD8 inhibits ferroptosis in pancreatic cancer by inhibiting the expression of RRAD. Cancer cell international 2023 — PMC10024404.
- PubMed PMID:8248782 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:9115241 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RRAD (GTP-binding protein RAD). Expected localisation Predominantly cytoplasmic; nuclear in a few tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A30715 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Respiratory epithelial cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human heart tissue; fixative not specified (datasheet A30715); 2. Fixation: Image fixative and duration unreported (datasheet A30715); 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 citrate buffer, pH 6.0 , 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-RRAD, 1:100 - 1:300 (datasheet A30715); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A30715); Caveat: Staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: One 1–308 chain; no isoforms or transmembrane segment (UniProt); Regulation: Higher in type II diabetic muscle (UniProt). Evidence: UniProt P55042, HPA tissue IHC · Uncertain, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet A30715, PMC6923381, PMC10024404. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RRAD/immunohistochemistry-ihc-guide.