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BAG6 / Large proline-rich protein BAG6 · IHC design guide
Design Immunohistochemistry for BAG6
Plan BAG6 staining in paraffin sections with 2–5 μg/mL of the IHC-validated antibody (datasheet A00967-1). Assess cytoplasmic and nuclear staining across tissues, using the high signal in spermatogonia as a reference (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
BAG6 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 and nuclear staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Widespread cytoplasmic/nuclear staining; high in spermatogonia (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A00967-1) | |
| Positive control | Lung+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 | Exosomal BAG6 may complicate tumor staining (UniProt) | |
| Regulation | Staining regulation is unestablished (UniProt) | |
| Isoform / epitope | 5 isoforms; epitope coverage is unverified (UniProt) |
Section 1
Recommended BAG6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol uses heat-mediated EDTA retrieval (datasheet A00967-1). Two published BAG6 IHC protocols add tissue-specific starting conditions (PMC6052567; PMC8748226).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A00967-1) |
| Fixation | Image fixative and duration unreported (datasheet A00967-1); 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 A00967-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00967-1) |
| Primary antibody | Rabbit anti-BAG6, 2-5μg/ml (datasheet A00967-1) |
| Primary incubation | Overnight at 4 °C (datasheet A00967-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A00967-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | BAG6-positive staining in alveolar cells type II of lung (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in most tissues, highly abundant in testis. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet A00967-1); the emitted article excerpts do not specify retrieval conditions (PMC6052567; PMC8748226).
Section 2
What Is the Expected BAG6 Staining Pattern?
BAG6 normally localizes to the cytosol and nucleus and has no transmembrane segment (UniProt P46379). In paraffin tissue sections, expect cytoplasmic and nuclear staining across many tissues, with particularly abundant staining in testis (HPA tissue IHC: Enhanced reliability). Lung alveolar type II cells, placental cytotrophoblasts, and testicular spermatogonia are reported high staining cell populations (HPA tissue IHC: High).
| Cytoplasmic and nuclear staining in identifiable alveolar type II cells, cytotrophoblasts, or spermatogonia. | This fits BAG6 localization (UniProt P46379) and the reported high staining in those cell populations (HPA tissue IHC: High). Judge intensity against cells within the same section; a strong signal alone does not establish specificity. |
| A sharply membrane restricted outline, with little nuclear or cytoplasmic signal. | That is discordant with the tissue pattern (HPA tissue IHC: cytoplasmic and nuclear) and BAG6 topology (UniProt P46379: no transmembrane segment). Check morphology and detection controls before assigning the outline to BAG6; an unexpected compartment can reflect an artefact. |
| Strong staining confined to a cell population outside the reported pattern, while expected cells remain unstained. | The distribution warrants a specificity check against the reported high and low cell populations (HPA tissue IHC). Cross-reactivity or endogenous detection activity are possibilities in chromogenic IHC (general IHC practice); morphology and a no-primary control help distinguish them. |
| Diffuse color across cells, stroma, and empty spaces without clear nuclear or cytoplasmic boundaries. | This does not reproduce the cellular tissue pattern (HPA tissue IHC: cytoplasmic and nuclear). Broad background can arise from detection reagents, inadequate blocking, or excess primary antibody (general IHC practice). Assess a no-primary control and clean tissue regions before scoring cells. |
| No convincing signal in an otherwise interpretable section containing reported high staining cells. | An absent signal in alveolar type II cells, cytotrophoblasts, or spermatogonia conflicts with their reported high staining (HPA tissue IHC: High). First verify that those cells are present, then review the antibody's IHC validation and the assay controls before calling the specimen BAG6 negative. |
💡Expected BAG6 appearanceCall a positive result when recognizable high staining cells show clear cytoplasmic and nuclear signal, especially spermatogonia, alveolar type II cells, or cytotrophoblasts (HPA tissue IHC: High; cytoplasmic and nuclear); diffuse color without cell boundaries or an isolated membrane outline is suspect (general IHC practice; UniProt P46379 topology).
| Evidence behind the tissue pattern | HPA rates BAG6 tissue IHC Enhanced, citing consistency between antibody staining and RNA expression (HPA tissue IHC). This supports a reference pattern, while interpretation of a new antibody and specimen still requires appropriate controls (general IHC practice). |
| Differences among tissues and cells | HPA reports high staining in selected lung, placental, and testicular cells, but low staining in oral squamous cells, skeletal myocytes, adipocytes, and other listed populations (HPA tissue IHC). Compare the same cell type and tissue context when judging intensity. |
| Isoforms and antibody epitope | UniProt lists 5 BAG6 isoforms (UniProt P46379). The supplied evidence gives no epitope for the catalog antibody, so isoform recognition and an isoform specific staining pattern cannot be predicted from this record. |
| Extracellular BAG6 | BAG6 can be released in exosomes by tumor and myeloid dendritic cells (UniProt P46379). This does not make extracellular chromogen an expected routine tissue IHC pattern; assess cellular localization and controls before interpreting extracellular deposits. |
| IF/ICC Q: What localization should I expect? | A: HPA reports mainly nucleoplasmic signal, with additional vesicular and cytosolic localization in ICC-IF (HPA subcellular: supported nucleoplasm and cytosol; approved vesicles). Interpret that evidence within IF/ICC; the tissue IHC reference remains cytoplasmic and nuclear (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known high staining cells show no chromogenic signal. | The relevant cells may be absent, or an assay step may have failed (HPA tissue IHC: High; general IHC practice). | Confirm cell identity, run a known-positive section, and check primary antibody, retrieval, and detection steps against the validated IHC procedure (general IHC practice). |
| Most of the section has diffuse color. | Excess primary antibody or insufficient blocking or washing can raise nonspecific background (general IHC practice). | Review the antibody's IHC dilution and blocking and washing steps; compare a no-primary control before scoring BAG6 (general IHC practice). |
| Color persists in a no-primary control. | Endogenous enzyme activity or nonspecific detection chemistry may contribute to chromogenic signal (general IHC practice). | Check the detection system's recommended endogenous activity block and reagent controls, then reassess tissue staining (general IHC practice). |
| Only cell membranes appear strongly outlined. | A membrane dominant pattern conflicts with reported localization (HPA tissue IHC: cytoplasmic and nuclear; UniProt P46379: no transmembrane segment). | Check tissue morphology, no-primary staining, and another IHC-validated antibody if available before accepting the compartment assignment (general IHC practice). |
| Low staining reference cells appear as strong as reported high staining cells. | The contrast disagrees with HPA's cell specific levels, though tissue context and assay conditions can affect visual comparisons (HPA tissue IHC; general IHC practice). | Verify cell identity and compare matched processing and detection controls; do not use a low staining population as an absolute negative control (HPA tissue IHC; general IHC practice). |
| A putative BAG6 signal varies unexpectedly between sections. | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. | Compare matched sections with the same validated IHC procedure and controls, then report unresolved variation without assigning it to a BAG6 specific fixation effect (general IHC practice). |
Experiment evidence
IHC & IF images showing BAG6 staining
A00967-1 · IHCIHC analysis of BAT3/BAG6 using anti-BAT3/BAG6 antibody (A00967-1). BAT3/BAG6 was detected in a paraffin-embedded section of human lung canc
A00967-1 · IHCIHC analysis of BAT3/BAG6 using anti-BAT3/BAG6 antibody (A00967-1). BAT3/BAG6 was detected in a paraffin-embedded section of human ovarian s
A00967-1 · IFIF analysis of BAT3/BAG6 using anti-BAT3/BAG6 antibody (A00967-1) and anti-Beta Tubulin antibody (M01857-3). BAT3/BAG6 was detected in immun
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000204463-BAG6/tissue/cerebral+cortex#imid_5476480
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000204463-BAG6/tissue/colon#imid_5476390
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000204463-BAG6/tissue/liver#imid_5476399
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000204463-BAG6/tissue/kidney#imid_5476392
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB020704; green = BAG6, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB020704; green = BAG6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for BAG6 IHC & IF
🧪Run lung first and confirm staining in alveolar type II cells (HPA: High in lung alveolar cells type II). HPA detects BAG6 in all 45 scored tissues, so no negative tissue is identified; use no-primary and isotype controls, and treat any cells on the positive slide as an internal negative reference only if they show counterstain without specific chromogen (HPA: no negative tissue rows; standard IHC control practice).
Positive control tissue: Lung (Alveolar cells type II, HPA High)
Negative control tissue: None in HPA: BAG6 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 BAG6 in A-431, U-251MG, U2OS, RPTEC/TERT1, hTERT-RPE1 (serum starved), NIH 3T3, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Run secondary-only and format-matched rabbit IgG isotype controls alongside BAG6 knockout material processed in parallel as a biological negative (A00967-1 tissue-IHC caption: rabbit primary; standard IHC control practice). Block endogenous peroxidase and inspect lung pigment or background before interpreting DAB staining (A00967-1 tissue-IHC caption: lung section and HRP/DAB detection; standard IHC practice).
⚠️Feasibility: The selected IHC-P caption reports heat retrieval in EDTA at pH 8.0, but its fixative is unreported; no target-specific fixation window or fixation effect is reported in the supplied evidence (A00967-1 tissue-IHC caption). The caption supports paraffin-section IHC after retrieval, while the supplied evidence does not establish whether frozen sections or IF are easier (A00967-1 tissue-IHC caption; HPA: ICC-IF images). Lung pigment and endogenous peroxidase can complicate chromogenic scoring, so compare the alveolar type II signal with the controls and local background (HPA: High in lung alveolar cells type II; standard IHC practice).
HPA tissue IHC evidence for BAG6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 |
|---|---|---|---|---|
| Lung | Alveolar cells type II | High | Protein (IHC) | HPA → |
| Placenta | Cytotrophoblasts | High | Protein (IHC) | HPA → |
| Testis | Spermatogonia cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: BAG6 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced BAG6 IHC Tips
Troubleshoot BAG6 staining in paraffin sections by checking retrieval, compartment, cell identity and detection controls before interpreting staining intensity.
Which retrieval conditions should I start with for BAG6 in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for the catalog antibody (datasheet A00967-1). The documented lung cancer paraffin-section image used this buffer, but its caption does not report a heating temperature or duration (datasheet A00967-1). If staining is weak, compare a small set of heating durations on adjacent sections while keeping the antibody concentration and detection steps constant (standard IHC practice). Check whether increased signal remains in recognizable cells and expected nuclear or cytoplasmic compartments, rather than appearing at section edges (HPA: cytoplasmic and nuclear tissue expression; standard IHC practice). Record retrieval conditions alongside each image so the comparison is reproducible (standard IHC practice).
How should I assess fixation as a cause of weak BAG6 staining?
Target-specific fixation sensitivity for BAG6 is unknown from the supplied IHC evidence (datasheet A00967-1: fixative not stated). The catalog image establishes staining in a paraffin-embedded lung cancer section, but it does not identify the fixative or fixation duration (datasheet A00967-1). When troubleshooting, compare sections with documented processing histories and hold EDTA retrieval at pH 8.0 constant (datasheet A00967-1; standard IHC practice). Examine nuclear detail, tissue preservation and staining gradients before attributing a weak result to BAG6 abundance (standard IHC practice). Do not infer fixation tolerance from its cytosolic and nuclear localization or reported phosphorylation sites (UniProt P46379; standard IHC practice).
Where should convincing BAG6 staining appear in tissue sections?
Assess both nuclear and cytoplasmic staining: the tissue profile reports expression in both compartments across most tissues (HPA: tissue IHC profile). Nucleoplasmic localization is supported by cell imaging, with additional cytosolic and vesicular localization reported there (HPA: subcellular profile). BAG6 has no transmembrane segment, so an exclusively crisp plasma-membrane outline needs independent validation before assignment to BAG6 (UniProt P46379 topology; standard IHC practice). Cytoplasmic retention can depend on GET4 interaction, making compartment shifts biologically plausible without proving their cause in a section (UniProt P46379). Score nuclear and cytoplasmic DAB separately against a counterstain, and inspect adjacent cells for consistent compartment boundaries (standard IHC practice).
Could epitope choice explain discordant BAG6 staining patterns?
BAG6 has 5 listed isoforms and an N-terminal ubiquitin-like domain spanning residues 17–92 (UniProt P46379). Its listed modifications include phosphorylation at residues 96, 113, 117 and 350 (UniProt P46379). The supplied catalog caption does not identify the antibody epitope, so it cannot establish which isoforms or modified forms the stain detects (datasheet A00967-1). Check the antibody's documented immunogen or epitope before comparing it with another reagent, and keep retrieval conditions matched when comparing adjacent sections (standard IHC practice). Treat discordant compartment staining as an epitope-dependent possibility until supported by an independently validated reagent or other evidence (standard IHC practice).
How can IF help resolve an ambiguous BAG6 IHC pattern?
Use a separate IF experiment to compare BAG6 with a marker for the cell population being assessed; alveolar type II cells and cytotrophoblasts have reported high tissue staining (HPA: tissue IHC profile). Choose a fluorophore whose emission can be distinguished from the specimen's autofluorescence, and include single-label and no-primary controls (standard IF practice). For an intracellular BAG6 epitope, use permeabilisation sufficient to admit antibody into the cytosol or nucleus, then check that compartment boundaries remain intact (UniProt P46379 localization; standard IF practice). Its catalog paraffin-section caption supports a chromogenic IHC workflow, with 2 μg/ml primary antibody overnight at 4°C; it does not establish an IF dilution (datasheet A00967-1).
What controls distinguish BAG6 signal from chromogenic background?
The documented IHC workflow used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and an anti-rabbit secondary for 30 minutes at 37°C (datasheet A00967-1). It used HRP with DAB for chromogenic development (datasheet A00967-1). Add a no-primary section and a peroxidase block to assess detection-system background; neither step is evidence of BAG6 specificity by itself (standard IHC practice). If diffuse DAB persists, check wash quality, secondary-only staining and chromogen development time on matched sections (standard IHC practice). Interpret granular deposits, edge accentuation and damaged areas against intact-cell nuclear and cytoplasmic patterns (HPA: tissue IHC profile; standard IHC practice).
How should I quantify BAG6 staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because BAG6 tissue staining is reported in both nuclei and cytoplasm (HPA: tissue IHC profile). For each compartment, report the percentage of positive cells and an H-score from staining intensity and the corresponding cell fractions (standard IHC practice). If counting positive cells per mm², normalize to the area of viable, evaluable tissue and state how excluded regions were selected (standard IHC practice). Keep retrieval, imaging settings and DAB development consistent across comparison groups (standard IHC practice). Use the same cell-type definition across samples; HPA reports different BAG6 staining levels among named cell populations (HPA: tissue IHC profile).
When should a BAG6-positive IHC result be considered credible?
A credible result shows staining in intact cells with interpretable nuclear or cytoplasmic boundaries, consistent with the reported tissue pattern (HPA: tissue IHC profile; standard IHC practice). Compare the stained cell type with the specimen annotation: high staining is reported in alveolar type II cells, cytotrophoblasts and spermatogonia (HPA: tissue IHC profile). An exclusively sharp membrane outline warrants scrutiny because BAG6 lacks a transmembrane segment (UniProt P46379 topology; standard IHC practice). Exclude necrotic regions and section edges from interpretation, and investigate staining retained on no-primary or peroxidase-control sections (standard IHC practice). Extracellular release in exosomes is reported, but diffuse extracellular DAB alone does not establish that mechanism (UniProt P46379; standard IHC practice).
Boster reagents
Best BAG6 / Large proline-rich protein BAG6 IHC Antibodies
A00967-1 has IHC images from human paraffin sections and an IF image from PC3 cells (catalog image captions). Its listed reactivity covers human, mouse, and rat (catalog applications/reactivity).
Real IHC data
Anti-BAT3/BAG6 Antibody ®
Cat # A00967-1
A00967-1 is the SKU that will render; its IHC images show human lung cancer and ovarian serous adenocarcinoma paraffin sections (catalog IHC captions). Its IF image shows PC3 cells, and its listed applications include IF and ICC (catalog IF caption; catalog applications).
Which to pick: Choose A00967-1 for tissue IHC in paraffin sections because its own IHC captions document those sections; the fixative is unreported (catalog IHC captions). Choose A00967-1 for IF/ICC because its own IF caption documents PC3 cells and ICC is a listed application (catalog IF caption; catalog applications). For human, mouse, or rat work, both A00967-1 and monoclonal M00967 list those species and IHC and IF applications, but only A00967-1 has supplied IHC and IF images (catalog reactivity/applications; catalog image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P46379 (BAG6_HUMAN, Large proline-rich protein BAG6).
- Human Protein Atlas. BAG6 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. BAG6 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to vesicles and cytosol..
- Human Protein Atlas. BAG6 antibody validation summary (3 antibodies).
- The dynamic changes in the number of uterine natural killer cells are specific to the eutopic but not to the ectopic endometrium in women and in a baboon model of endometriosis. Reproductive biology and endocrinology : RB&E 2018 — PMC6052567.
- Nodular lymphocyte predominant hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma--endpoints of a spectrum of one disease?. PloS one 2013 — PMC3823948.
- GET4 is a novel driver gene in colorectal cancer that regulates the localization of BAG6, a nucleocytoplasmic shuttling protein. Cancer science 2022 — PMC8748226.
- SGTA associates with intracellular aggregates in neurodegenerative diseases. Molecular brain 2021 — PMC7986274.
- PubMed PMID:2156268 — UniProt-cited evidence.
- PubMed PMID:17974005 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for BAG6 (Large proline-rich protein BAG6). Expected localisation Cytoplasmic and nuclear staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A00967-1). Validated IHC antibody: A00967-1 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Lung (Alveolar cells type II); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A00967-1); 2. Fixation: Image fixative and duration unreported (datasheet A00967-1); 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 A00967-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00967-1); 8. Primary antibody: Rabbit anti-BAG6, 2-5μg/ml (datasheet A00967-1); 9. Primary incubation: Overnight at 4 °C (datasheet A00967-1). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Exosomal BAG6 may complicate tumor staining (UniProt); Isoform / epitope: 5 isoforms; epitope coverage is unverified (UniProt); Regulation: Staining regulation is unestablished (UniProt). Evidence: UniProt P46379, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to…, Datasheet A00967-1, PMC6052567, PMC8748226. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/BAG6/immunohistochemistry-ihc-guide.