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PA2G4 / Proliferation-associated protein 2G4 · IHC design guide
Design Immunohistochemistry for PA2G4
Use colon glandular cells as a high-staining reference (HPA tissue IHC) and start the catalog antibody at 0.5–1 µg/ml (datasheet A02791). Score cytoplasmic staining separately from nuclear signal: tissue IHC shows a cytoplasmic profile (HPA tissue IHC), while HRG can promote nuclear translocation (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PA2G4 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Widespread cytoplasmic staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A02791) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific) | |
| Caveat | HRG can shift PA2G4 toward the nucleus (UniProt) | |
| Regulation | Isoform 1 is strong in cancer cells (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unreported (UniProt) |
Section 1
Recommended PA2G4 IHC & IF Protocols
Compare the catalog antibody’s citrate pH 6 IHC-P protocol (datasheet A02791) with a published NPC and NP paraffin-section protocol (PMC8354561 methods).
| Sample | Paraffin-embedded rat intestine tissues; fixative not specified (datasheet A02791) |
| Fixation | Image fixative and duration unreported (datasheet A02791); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A02791) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02791) |
| Primary antibody | Rabbit anti-PA2G4, 0.5-1μg/ml (datasheet A02791) |
| Primary incubation | Overnight at 4 °C (datasheet A02791) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A02791) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | PA2G4-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval (datasheet A02791); the published protocol specifies 10 mM citrate at 100°C for 2 minutes (PMC8354561 methods).
Section 2
What Is the Expected PA2G4 Staining Pattern?
PA2G4 staining should be predominantly cytoplasmic in many cell types, including glandular cells in colon and duodenum and respiratory epithelial cells in bronchus (HPA: ubiquitous cytoplasmic expression; High in these cells). HPA rates tissue IHC reliability as Supported, with medium agreement between staining and RNA data (HPA: tissue IHC). Nuclear and nucleolar localization is also described (UniProt Q9UQ80: subcellular location). PA2G4 has no transmembrane segment (UniProt Q9UQ80: topology).
| Clear cytoplasmic stain in colon or duodenal glandular cells (HPA: High in both). | This fits the reported tissue pattern (HPA: ubiquitous cytoplasmic expression). Judge a positive result by cellular localization as well as intensity; chromogen outside recognizable cells does not establish PA2G4 staining (general IHC practice). |
| Predominantly membrane, extracellular, or tissue-edge color. | These are unexpected as the principal PA2G4 pattern: UniProt reports cytoplasmic, nuclear, and nucleolar locations and no transmembrane segment (UniProt Q9UQ80: subcellular location; topology). Check for deposits or edge effects (general IHC practice). |
| Strong staining in skeletal myocytes or smooth muscle cells. | HPA reports Low staining in these cell types (HPA: skeletal muscle myocytes; smooth muscle cells). Recheck cell identification and compare with a reported High cell population before interpreting strong color as PA2G4; cross-reactivity or endogenous detection activity is possible (general IHC practice). |
| Uniform faint color across cells, stroma, and empty spaces. | A pattern that ignores cell boundaries is difficult to score against HPA’s cellular cytoplasmic pattern (HPA: tissue IHC profile). Consider nonspecific background or detection reagent activity and inspect a no-primary control (general IHC practice). |
| No cytoplasmic signal in colon glandular cells or bronchial respiratory epithelium. | Both are reported High by HPA (HPA: colon glandular cells; bronchus respiratory epithelial cells). Confirm tissue identity and control performance, then review the validated IHC procedure; a blank slide alone cannot establish absent PA2G4 (general IHC practice). |
💡Expected PA2G4 appearanceCall a result positive when recognizable glandular or respiratory epithelial cells show clear cytoplasmic staining in an HPA High tissue; diffuse extracellular color or dominant membrane staining is suspect (HPA: tissue IHC profile; UniProt Q9UQ80: topology; general IHC practice).
| Tissue and cell selection (HPA: tissue IHC). | HPA reports High staining in colon and duodenal glandular cells and bronchial respiratory epithelial cells, but Low staining in skeletal myocytes and smooth muscle cells (HPA: tissue IHC). Compare identified cells, not whole-section color (general IHC practice). |
| Strength of tissue evidence (HPA: reliability Supported). | HPA calls the tissue pattern Supported and describes medium consistency with RNA expression (HPA: tissue IHC). Treat the reported pattern as a guide for interpretation, while retaining slide controls and cell-level assessment (general IHC practice). |
| Compartment and signaling context (UniProt Q9UQ80: subcellular location). | UniProt lists cytoplasm and nucleus, including nucleolus, and reports nuclear translocation after HRG treatment (UniProt Q9UQ80: subcellular location). Nuclear color therefore needs context; it should not automatically be scored as an artifact or as the expected dominant tissue pattern. |
| Nucleolar regulation (UniProt Q9UQ80: Ser-361). | Phosphorylation at Ser-361 by PKC/PRKCD regulates nucleolar localization (UniProt Q9UQ80: modified residues; subcellular location). This supports biological variation in compartment, but supplies no predicted staining intensity for a particular paraffin section. |
| Isoforms (UniProt Q9UQ80: isoforms 1 and 2). | Both isoforms are widely expressed; at protein level, isoform 1 is strongly expressed in cancer cells while isoform 2 is undetectable there (UniProt Q9UQ80: tissue specificity). The supplied evidence does not establish whether a given IHC antibody distinguishes them. |
| IF/ICC Q: What compartment is reported? (HPA: subcellular ICC-IF). | A: HPA reports supported cytosolic localization in ICC-IF (HPA: subcellular ICC-IF). That observation helps interpret compartment, but does not specify a paraffin IHC procedure or override UniProt’s context-dependent nuclear localization (UniProt Q9UQ80: subcellular location). |
| Situation | Likely cause | Next action |
|---|---|---|
| Reported High tissue is blank (HPA: colon glandular cells; bronchial respiratory epithelial cells). | The tissue or target cell may be misidentified, or an IHC workflow step may have failed (general IHC practice). | Confirm the cell population, compare a known-positive section, and review the IHC-validated antibody’s supplied procedure and detection controls (general IHC practice). |
| Only nuclei stain in an untreated comparison section. | Nuclear PA2G4 is biologically possible, but HPA describes the tissue pattern as cytoplasmic (UniProt Q9UQ80: subcellular location; HPA: tissue IHC profile). | Check for cytoplasmic signal in a reported High cell population and compare controls; interpret nuclear enrichment alongside experimental context (HPA: tissue IHC; UniProt Q9UQ80: HRG translocation; general IHC practice). |
| Strong color appears in myocytes despite weak staining in glandular controls. | HPA reports Low staining in skeletal myocytes, while colon and duodenal glandular cells are High (HPA: tissue IHC). Cell identification or assay specificity may need review (general IHC practice). | Reidentify the cells, inspect no-primary controls for endogenous detection activity, and compare staining with a reported High tissue (HPA: tissue IHC; general IHC practice). |
| Color coats stroma, lumens, or tissue edges. | This distribution does not match HPA’s cellular cytoplasmic profile; precipitate, uneven washing, or edge effects are possible (HPA: tissue IHC profile; general IHC practice). | Inspect the no-primary control and slide surface; review reagent preparation and washing before scoring cells (general IHC practice). |
| Cytoplasmic color is too faint to distinguish from background. | Low-expressing cells or insufficient assay sensitivity are possibilities; HPA reports Low staining in skeletal myocytes and smooth muscle cells (HPA: tissue IHC; general IHC practice). | Compare a reported High cell population on a suitable control section, then assess signal against background using the antibody’s IHC guidance (HPA: tissue IHC; general IHC practice). |
| A nuclear or nucleolar pattern changes between experimental conditions. | PA2G4 can translocate to the nucleus after HRG treatment, and Ser-361 phosphorylation regulates nucleolar localization (UniProt Q9UQ80: subcellular location). | Record treatment and compartment-specific scores, then compare matched sections and controls before assigning a technical cause (UniProt Q9UQ80: subcellular location; general IHC practice). |
Experiment evidence
IHC & IF images showing PA2G4 staining
A02791 · IHCIHC analysis of EBP1 using anti-EBP1 antibody (A02791). EBP1 was detected in paraffin-embedded section of rat intestine tissues. Heat mediat
A02791 · IHCIHC analysis of EBP1 using anti-EBP1 antibody (A02791). EBP1 was detected in paraffin-embedded section of mouse intestine tissues. Heat medi
A02791-1 · IHCFormalin-fixed and paraffin-embedded human lung carcinoma reacted with PA2G4 Antibody (Center R243), which was peroxidase-conjugated to the
A02791-1 · IFFluorescent confocal image of U251 cell stained with PA2G4 Antibody (Center R243).U251 cells were fixed with 4% PFA (20 min), permeabilized
A02791-2 · IHCEBP1 Antibody (C-term) IHC analysis in formalin fixed and paraffin embedded mouse brain tissue followed by peroxidase conjugation of the sec
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000170515-PA2G4/tissue/cerebral+cortex#imid_3928672
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000170515-PA2G4/tissue/colon#imid_3928598
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000170515-PA2G4/tissue/liver#imid_3928591
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000170515-PA2G4/tissue/kidney#imid_3928584
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB011711; green = PA2G4, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB011711; green = PA2G4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PA2G4 IHC & IF
🧪Run appendix first: its glandular cells must show PA2G4 staining (HPA: High in appendix glandular cells). HPA detects PA2G4 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat cells without staining above background on the positive slide as internal negatives only if observed (HPA: no negative tissue rows).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: None in HPA: PA2G4 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 PA2G4 in A-431, U2OS, U-251MG, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide, a concentration-matched rabbit isotype control, and, where available, PA2G4-knockout material as a biological negative (caption: rabbit primary; standard IHC practice). For chromogenic detection, quench endogenous peroxidase and assess endogenous biotin background when using the caption’s streptavidin–biotin system (caption: SABC with DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A02791 paraffin-section caption does not state a fixative (caption: fixative unreported). The caption demonstrates staining after heat retrieval in citrate buffer at pH 6 for 20 minutes, but does not establish that retrieval is required (caption: antigen retrieval conditions). The evidence does not establish whether frozen sections or IF are easier; assess biotin-related background in appendix glandular tissue when using the caption’s SABC detection (caption: SABC; standard IHC practice).
HPA tissue IHC evidence for PA2G4
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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal 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: PA2G4 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced PA2G4 IHC Tips
Troubleshoot PA2G4 staining in paraffin section IHC by checking retrieval, compartment patterns, controls and cell specific scoring.
Which retrieval conditions should I try first for PA2G4 in paraffin sections?
Start with heat mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet A02791). That condition was used for PA2G4 staining in a paraffin section of rat intestine, so use the same staining run to compare any later changes (datasheet A02791; standard IHC practice). If staining is weak, vary heating duration on matched sections while holding antibody concentration and detection constant, and watch for damaged tissue or rising background (standard IHC practice). A weak result alone cannot establish retrieval failure: the documented example also used 1 μg/ml primary antibody overnight at 4°C (datasheet A02791).
Could fixation explain weak or uneven PA2G4 staining?
Target specific fixation sensitivity is unknown because the selected paraffin section caption does not state a fixative (datasheet A02791). Record the fixative and fixation duration for each specimen, then compare sections processed together before attributing intensity differences to PA2G4 biology (standard IHC practice). Uneven staining near tissue edges, folds or poorly preserved areas warrants a check of section quality and processing history (standard IHC practice). Keep citrate retrieval at pH 6 for 20 minutes and the primary antibody at 1 μg/ml during that comparison, matching the documented staining example (datasheet A02791).
How should cytoplasmic and nuclear PA2G4 staining be judged?
Expect a predominantly cytoplasmic tissue pattern because the tissue IHC profile reports ubiquitous cytoplasmic expression (HPA tissue IHC). Cytosolic localisation also has support from cell imaging, while UniProt lists cytoplasm and nucleolus and describes nuclear translocation after HRG treatment (HPA subcellular; UniProt Q9UQ80 subcellular). Score cytoplasmic and nuclear signal separately, using the same counterstain and compartment definitions across specimens (standard IHC practice). A nuclear or nucleolar pattern needs appropriate morphology and controls before biological interpretation; phosphorylation at Ser-361 regulates nucleolar localisation, but a chromogenic section cannot establish that modification by itself (UniProt Q9UQ80 modified residues; standard IHC practice).
Can this stain distinguish PA2G4 isoforms or phosphorylation states?
Do not assign an isoform from staining intensity unless the antibody epitope and its isoform coverage are established (standard IHC practice). PA2G4 has 2 annotated isoforms, and the supplied antibody caption does not identify an isoform specific epitope (UniProt Q9UQ80 isoforms; datasheet A02791). Ser-361 phosphorylation regulates nucleolar localisation, so a changed nuclear pattern does not demonstrate recognition of phosphorylated PA2G4 (UniProt Q9UQ80 modified residues; standard IHC practice). Compare antibody documentation with the isoform sequences and test suitable controls before making an isoform or modification claim; retain separate cytoplasmic and nuclear scores in the meantime (standard IHC practice).
How can IF help check a PA2G4 IHC localisation result?
For a separate IF assay, multiplex PA2G4 with a marker that identifies the expected glandular cell population, such as the glandular cells reported with high tissue IHC staining in colon (HPA tissue IHC; standard IF practice). Choose a fluorophore outside the strongest tissue autofluorescence and inspect single colour controls before interpreting overlap (standard IF practice). PA2G4 has no annotated transmembrane segment and is reported in the cytosol and nucleolus, so assess permeabilisation for intracellular epitope access without assuming which epitope the antibody recognises (UniProt Q9UQ80 topology and subcellular; standard IF practice). Compare compartments with the IHC result, while treating each assay’s fixation and detection conditions separately (standard IF practice).
What should I check when PA2G4 DAB staining looks diffuse?
First compare the stained section with a no primary control and examine whether colour follows tissue edges, damaged regions or expected cellular compartments (standard IHC practice). The documented rat intestine example used 10% goat serum blocking, a biotinylated secondary system and DAB, making nonspecific binding and detection background useful checks for that workflow (datasheet A02791; standard IHC practice). Check endogenous peroxidase blocking and, with a biotin based system, an appropriate detection control before assigning diffuse brown signal to PA2G4 (standard IHC practice). Retitrate the documented 1 μg/ml primary concentration only after checking controls and keeping retrieval constant (datasheet A02791; standard IHC practice).
How should PA2G4 staining be scored across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because PA2G4 tissue IHC is described as ubiquitously cytoplasmic whereas nuclear localisation is also recorded (HPA tissue IHC; UniProt Q9UQ80 subcellular). Record the percentage of positive cells and an intensity based H-score separately for cytoplasm and nucleus, using consistent thresholds and a common counterstain (standard IHC practice). Normalise positive cell counts to the number of evaluable cells in that population, or report positive cell density per mm² of evaluable tissue when cell counts are impractical (standard IHC practice). Exclude necrosis, folds and edge artefacts consistently, and compare batches using reference sections processed together (standard IHC practice).
When is an apparent PA2G4 positive result convincing?
A convincing result shows cellular staining in the compartment being scored and persists under appropriate detection controls (standard IHC practice). Cytoplasmic staining fits the supported tissue profile, and high staining in colon glandular cells offers a documented cell population for comparison; cholangiocytes are reported at low levels (HPA tissue IHC). Nuclear or nucleolar signal can be biologically plausible, but assess it separately because UniProt records those locations and HRG responsive nuclear translocation (UniProt Q9UQ80 subcellular). Discount colour confined to tissue edges, necrotic areas or structures positive in a no primary control; check endogenous enzyme activity before calling DAB deposits PA2G4 (standard IHC practice).
Boster reagents
Best PA2G4 / Proliferation-associated protein 2G4 IHC Antibodies
Anti-PA2G4 antibodies have IHC images from rat and mouse tissues and human carcinomas (catalog IHC captions), plus an IF image from U251 cells (A02791-1 IF caption).
Real IHC data
Anti-EBP1/PA2G4 Antibody ®
Cat # A02791
Real IHC data
Anti-PA2G4 Antibody (Center R243)
Cat # A02791-1
Real IHC data
Anti-EBP1 Antibody (C-term)
Cat # A02791-2
A02791 has paraffin-section IHC images from rat intestine, mouse intestine and brain, and human stomach cancer; its listed reactivity is human, mouse and rat (A02791 IHC captions; catalog reactivity). A02791-1 has an IHC image from formalin-fixed, paraffin-embedded human lung carcinoma, while A02791-2 has one from formalin-fixed, paraffin-embedded mouse brain (A02791-1 and A02791-2 IHC captions).
Which to pick: For paraffin-section, paraffin-embedded tissue IHC, choose A02791-1 for human samples or A02791-2 for mouse samples; both list IHC-P and are polyclonal (catalog applications and dilution entries; respective IHC captions). For IF/ICC, choose A02791-1: it lists IF and has an image from PFA-fixed, permeabilized U251 cells (A02791-1 catalog applications; IF caption). For work across human, mouse and rat, consider A02791, which lists reactivity with all three and has paraffin-section IHC images; its image captions do not report a fixative (A02791 catalog reactivity; IHC captions). The selected A02791 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A02791).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UQ80 (PA2G4_HUMAN, Proliferation-associated protein 2G4).
- Human Protein Atlas. PA2G4 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. PA2G4 subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. PA2G4 antibody validation summary (3 antibodies).
- Increased PA2G4 Expression Is an Unfavorable Factor in Nasopharyngeal Carcinoma. Applied immunohistochemistry & molecular morphology : AIMM 2021 — PMC8354561.
- PA2G4 promotes the metastasis of hepatocellular carcinoma by stabilizing FYN mRNA in a YTHDF2-dependent manner. Cell & bioscience 2022 — PMC9080163.
- Evidence for a fragile X messenger ribonucleoprotein 1 (FMR1) mRNA gain-of-function toxicity mechanism contributing to the pathogenesis of fragile X-associated premature ovarian insufficiency. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2022 — PMC9828574.
- PA2G4 Functions as a Cofactor for MYC Family Oncoproteins in MYC-Driven Malignancies. Cells 2025 — PMC12468391.
- PubMed PMID:9345902 — UniProt-cited evidence.
- PubMed PMID:10682683 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PA2G4 (Proliferation-associated protein 2G4). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A02791). Validated IHC antibody: A02791 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (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 rat intestine tissues; fixative not specified (datasheet A02791); 2. Fixation: Image fixative and duration unreported (datasheet A02791); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: Citrate pH 6 , 20 min (datasheet A02791); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A02791); 8. Primary antibody: Rabbit anti-PA2G4, 0.5-1μg/ml (datasheet A02791); 9. Primary incubation: Overnight at 4 °C (datasheet A02791). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: HRG can shift PA2G4 toward the nucleus (UniProt); Isoform / epitope: 2 isoforms; epitope coverage is unreported (UniProt); Regulation: Isoform 1 is strong in cancer cells (UniProt). Evidence: UniProt Q9UQ80, HPA tissue IHC · Supported, HPA ICC-IF · cytosol., Datasheet A02791, PMC8354561. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PA2G4/immunohistochemistry-ihc-guide.