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CHAC1 / Glutathione-specific gamma-glutamylcyclotransferase 1 · IHC design guide
Design Immunohistochemistry for CHAC1
Plan CHAC1 paraffin-section IHC using the catalog antibody’s documented pancreas staining workflow (datasheet A09017-2). Compare cytoplasmic staining with HPA tissue IHC, including pancreatic endocrine cells and kidney tubule cells, while accounting for its low consistency with RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CHAC1 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 | General cytoplasmic staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining across multiple tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A09017-2) | |
| 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 (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 A09017-2) | |
| Caveat | Staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | UPR-linked; intensity effect unproven (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope overlap is unknown (UniProt) |
Section 1
Recommended CHAC1 IHC & IF Protocols
The catalog antibody protocol uses EDTA pH 8.0 retrieval (datasheet A09017-2). The published IHC protocols below provide conditions reported for gastric mucosa, kidney carcinoma, and lung adenocarcinoma samples (PMC6618068; PMC8034464; PMC11455913).
| Sample | Paraffin-embedded human pancreas tissue; fixative not specified (datasheet A09017-2) |
| Fixation | Image fixative and duration unreported (datasheet A09017-2); 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 A09017-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A09017-2) |
| Primary antibody | Rabbit anti-CHAC1, 1:50 recommended; image 2 μg/ml (datasheet A09017-2) |
| Primary incubation | Overnight at 4 °C (datasheet A09017-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A09017-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CHAC1-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 for the catalog antibody (datasheet A09017-2). For the published gastric-mucosa antibody, the reported alternative is citrate pH 6.0 (PMC6618068).
Section 2
What Is the Expected CHAC1 Staining Pattern?
CHAC1 is a soluble protein annotated in the cytosol and trans-Golgi network, with no transmembrane segment (UniProt Q9BUX1). In paraffin-section IHC, expect predominantly cytoplasmic staining in selected glandular, neuronal, renal tubular and pancreatic endocrine cells (HPA: general cytoplasmic expression; listed cells High). Treat intensity as a tissue-level guide: HPA rates its IHC evidence Approved but reports low consistency between antibody staining and RNA expression (HPA: reliability description).
| Cytoplasmic staining in appendix or gallbladder glandular cells, with less color in neighboring structures. | This matches the reported cell distribution and general cytoplasmic profile (HPA: Appendix and Gallbladder glandular cells High; general cytoplasmic expression). Compare cells within the same section before judging absolute color intensity; local tissue handling and chromogen development can affect that comparison (general IHC practice). |
| Cytoplasmic signal in kidney tubules, pancreatic endocrine cells, or Purkinje cells. | These are supported positive patterns (HPA: kidney cells in tubules High; pancreatic endocrine cells High; cerebellar Purkinje cells High). Record which cell population stains. A whole-tissue average can obscure a strong signal restricted to the listed cells (general IHC interpretation). |
| Predominantly nuclear or extracellular deposit instead of a cell-associated cytoplasmic pattern. | Neither compartment is the reported IHC pattern (HPA: general cytoplasmic expression; UniProt Q9BUX1: cytosol and trans-Golgi network). Review morphology, counterstain and the negative control before assigning this deposit to CHAC1; misplaced chromogen alone does not establish a second localization (general IHC practice). |
| Strong staining centered on an unlisted cell population while the expected cells are weak. | Consider cross-reactivity or endogenous detection activity, especially if the control shows the same distribution (general IHC practice). HPA lists specific High cell populations and reports low antibody–RNA consistency; its list cannot establish that every unlisted cell is truly negative (HPA: tissue IHC profile and reliability description). |
| Diffuse color across cells and stroma, or no signal in an HPA High cell population. | Diffuse deposit limits localization and suggests a background problem (general IHC practice). An absent signal merits a run-control check before a biological conclusion; HPA High describes observed staining, not guaranteed positivity in every specimen (HPA: tissue IHC profile; Approved with low antibody–RNA consistency). |
💡Expected CHAC1 appearanceCall a convincing IHC positive a cell-associated, predominantly cytoplasmic signal in an HPA High population, such as kidney tubular cells; conspicuous nuclear, extracellular or diffuse stromal color is suspect (HPA: general cytoplasmic expression; kidney cells in tubules High; UniProt Q9BUX1: cytosol and trans-Golgi network).
| Cell selection for interpretation | HPA reports High staining in selected glandular, neuronal, tubular and pancreatic endocrine cells; interpret those populations rather than labeling an entire organ uniformly positive (HPA: tissue IHC profile). |
| IHC evidence strength | The tissue profile is Approved, with low consistency between antibody staining and RNA expression; use the images as pattern guidance and avoid treating RNA abundance as a staining-intensity prediction (HPA: reliability description). |
| RNA versus protein example | Skeletal muscle has tissue-enhanced RNA specificity yet Low staining in myocytes. Choose a cell-level IHC control from the High staining list instead of inferring protein staining from RNA alone (HPA: RNA specificity; skeletal muscle myocytes Low). |
| Topology and processing | CHAC1 has no transmembrane segment, signal peptide, annotated propeptide or glycosylation site; UniProt lists the chain as residues 1–222. These annotations support a cell-associated interpretation but do not identify an antibody epitope or predict antigen-retrieval behavior (UniProt Q9BUX1). |
| IF/ICC Q&A: should mitochondria be expected in IHC? | HPA approves a mitochondrial ICC-IF location, whereas its tissue IHC profile is generally cytoplasmic and UniProt lists cytosol and trans-Golgi network. Report this assay-dependent difference; mitochondrial ICC-IF images do not establish mitochondrial resolution in chromogenic tissue IHC (HPA: subcellular and tissue profiles; UniProt Q9BUX1). |
| Situation | Likely cause | Next action |
|---|---|---|
| No visible staining in kidney tubules or appendix glandular cells. | The run may have a detection or tissue-handling failure; an absent signal alone cannot distinguish technical failure from specimen variation (general IHC practice; HPA: these cell populations High). | Check a same-run positive-control section, reagent sequence and chromogen development. If the run control works, review morphology and the specific cell population before recording a negative result (general IHC practice). |
| Color covers stroma and cells without clear borders. | Background may arise from nonspecific reagent binding or excessive chromogen development (general IHC practice). | Inspect the reagent-omission control, blocking and wash steps; compare background with a positive cell population on the same run (general IHC practice; HPA: general cytoplasmic expression). |
| Nuclear color dominates the expected cytoplasmic signal. | The distribution conflicts with reported tissue IHC and UniProt localization; counterstain or nonspecific deposit may complicate reading (HPA: general cytoplasmic expression; UniProt Q9BUX1). | Compare the chromogen color with the nuclear counterstain and controls. Score CHAC1 only where a distinct cell-associated pattern is credible (general IHC practice). |
| Unlisted cells stain more strongly than the expected population. | Cross-reactivity or endogenous detection activity is possible, while an unlisted cell is not automatically CHAC1-negative (general IHC practice; HPA: listed High populations). | Check a detection-reagent control and examine a second HPA High cell population. Describe the unexpected distribution separately rather than calling it validated CHAC1 staining (general IHC practice; HPA: tissue IHC profile). |
| Skeletal muscle looks weak despite tissue-enhanced RNA. | The observations concern different measurements: HPA reports Low staining in myocytes and tissue-enhanced skeletal-muscle RNA (HPA: tissue IHC and RNA specificity). | Do not use skeletal muscle as the sole positive IHC control. Compare the run with an HPA High cell population and document the RNA–staining discrepancy (HPA: tissue IHC profile and reliability description). |
| An IF image looks mitochondrial, but chromogenic IHC looks broadly cytoplasmic. | The assays have different reported patterns and spatial resolution; HPA approves mitochondrial ICC-IF localization and describes general cytoplasmic tissue IHC (HPA: subcellular and tissue profiles). | Use the tissue IHC profile to score paraffin sections. Keep the ICC-IF observation assay-specific and avoid claiming mitochondrial IHC localization from diffuse chromogen (HPA: subcellular and tissue profiles; general IHC interpretation). |
Experiment evidence
IHC & IF images showing CHAC1 staining
A09017-2 · IHCIHC analysis of CHAC1 using anti-CHAC1 antibody (A09017-2). CHAC1 was detected in a paraffin-embedded section of human pancreas tissue. Heat
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000128965-CHAC1/tissue/cerebral+cortex#imid_11166360
HPA · Thyroid glandHuman Protein Atlas IHC, Thyroid gland. Source: https://www.proteinatlas.org/ENSG00000128965-CHAC1/tissue/thyroid+gland#imid_11166320
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000128965-CHAC1/tissue/colon#imid_11166286
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000128965-CHAC1/tissue/liver#imid_11166287
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042708; green = CHAC1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042708; green = CHAC1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CHAC1 IHC & IF
🧪Run appendix first and look for staining in glandular cells (HPA: High in appendix glandular cells). HPA detects CHAC1 in all 45 scored tissues and lists no negative tissue, so no-primary and isotype controls carry the negative comparison; no appendix cell type is established as an internal negative, and cells without visible signal should be treated only as a local background reference (HPA: no negative tissue or appendix cell type supplied).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: None in HPA: CHAC1 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 CHAC1 in A-431, U-251MG, U2OS, with annotated localisation: Mitochondria (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide, a concentration-matched rabbit IgG isotype control, and CHAC1-knockout tissue or cells as a biological negative (caption: rabbit primary antibody; standard IHC controls). Quench endogenous peroxidase and check for background before interpreting appendix staining with HRP/DAB (caption: HRP/DAB detection; standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported; the selected A09017-2 paraffin-section caption does not state its fixative (selected-SKU tissue-IHC caption: fixative not stated). The caption reports heat retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required or whether frozen sections or IF would be easier (selected-SKU tissue-IHC caption). Endogenous peroxidase is a potential artefact with HRP/DAB detection in appendix sections (caption: HRP/DAB detection; standard chromogenic IHC practice).
HPA tissue IHC evidence for CHAC1
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 | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: CHAC1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced CHAC1 IHC Tips
Use the catalog antibody’s paraffin-section result as a starting point, then validate cell type, compartment and background in each IHC experiment.
Which antigen retrieval should I start with for CHAC1 in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A09017-2). The selected paraffin-section image used this retrieval before overnight incubation at 4°C with 2 μg/ml antibody, so retain those conditions while assessing retrieval (datasheet A09017-2). If staining is weak, compare a second retrieval condition on adjacent sections while keeping antibody concentration, detection and development constant (standard IHC practice). Judge improvement by signal in the expected cells and by background, using pancreatic endocrine cells as one reference population (HPA: High in pancreatic endocrine cells). Record heating and cooling conditions because changes in either can alter epitope exposure (standard IHC practice).
How should I troubleshoot CHAC1 staining when fixation history is uncertain?
The selected image shows a paraffin-embedded pancreas section, but its caption does not report the fixative; CHAC1-specific fixation sensitivity is therefore unknown (datasheet A09017-2). Record the fixative and fixation duration for each specimen, and compare sections with known processing histories when signal differs (standard IHC practice). Begin with EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody, the documented conditions for the selected image (datasheet A09017-2). Assess morphology alongside staining, since damaged tissue and uneven processing can mislead intensity comparisons (standard IHC practice). Keep processing groups separate during scoring until comparable staining is demonstrated (standard IHC practice).
What CHAC1 staining pattern should I accept in tissue sections?
Expect predominantly cytoplasmic staining as the tissue-level reference pattern (HPA: general cytoplasmic expression). UniProt places CHAC1 in the cytosol and trans-Golgi network, while an approved ICC/IF record places it in mitochondria; these sources support checking compartment calls across methods (UniProt Q9BUX1 subcellular location; HPA: mitochondria approved). In chromogenic sections, inspect whether signal belongs to intact cells and whether its distribution is reproducible across adjacent sections (standard IHC practice). Strong nuclear-only or extracellular staining needs independent validation before assignment to CHAC1 (UniProt Q9BUX1 subcellular location). Use cell morphology and a matched negative control to assess ambiguous puncta (standard IHC practice).
Can this antibody distinguish CHAC1 isoforms or a modified epitope?
CHAC1 has 2 listed isoforms, but the supplied records do not map the catalog antibody’s epitope or establish isoform selectivity (UniProt Q9BUX1 isoforms; datasheet A09017-2). Its annotated chain spans residues 1–222, with no annotated glycosylation or modified residues (UniProt Q9BUX1 processing and PTMs). Treat a positive chromogenic signal as CHAC1 staining without assigning it to a particular isoform or modification (UniProt Q9BUX1 isoforms; datasheet A09017-2). If that distinction matters, obtain an epitope map and test isoform-specific controls before interpreting section staining (standard IHC validation practice). Compare controls under identical retrieval and detection conditions (standard IHC practice).
How can I compare CHAC1 immunofluorescence with this chromogenic IHC result?
Treat IF/ICC as a separate validation experiment: the selected product caption documents paraffin-section chromogenic IHC, while HPA reports approved mitochondrial ICC/IF localisation (datasheet A09017-2; HPA: mitochondria approved). Multiplex CHAC1 with a validated marker for the expected cell population, such as pancreatic endocrine cells when examining pancreas (HPA: High in pancreatic endocrine cells; standard IF practice). Select a fluorophore channel with low tissue autofluorescence and include single-label and no-primary controls to check bleed-through and background (standard IF practice). Permeabilise sufficiently for antibody access to cytosolic and Golgi-associated epitopes; verify access separately if testing mitochondrial localisation (UniProt Q9BUX1 subcellular location; HPA: mitochondria approved; standard IF practice). Do not infer IF fixation conditions from the paraffin-section caption (datasheet A09017-2).
What should I check when CHAC1 DAB staining appears diffuse?
The selected image used 10% goat serum blocking, a peroxidase-conjugated secondary and DAB development (datasheet A09017-2). Include a no-primary section, block endogenous peroxidase before detection, and compare background with the full staining run (standard chromogenic IHC practice). If diffuse color persists, check secondary binding, incomplete washing and excess chromogen development while holding retrieval at EDTA pH 8.0 (standard IHC practice; datasheet A09017-2). Separate pigment or damaged-tissue color from cellular signal by examining morphology and the no-primary control (standard IHC practice). Accept a CHAC1 call only when the expected cellular distribution exceeds those controls (HPA: general cytoplasmic expression; standard IHC validation practice).
How should I score CHAC1 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, then use percent positive cells or an H-score for cellular DAB intensity (standard IHC quantification practice). For an H-score, record the percentages at intensities 0–3 and sum percentage times intensity, yielding a 0–300 scale (standard IHC quantification practice). Normalise counts or stained area to viable target cells or tissue area, reported per mm², and exclude necrosis and section edges consistently (standard IHC quantification practice). Score pancreatic endocrine cells separately if using the documented pancreas reference (HPA: High in pancreatic endocrine cells). Keep retrieval, antibody exposure, imaging and scoring thresholds constant across comparisons (datasheet A09017-2; standard IHC practice).
How do I distinguish true CHAC1 signal from staining artefact?
A credible result is reproducible cellular staining in an expected population, such as pancreatic endocrine cells, with a predominantly cytoplasmic tissue pattern (HPA: High in pancreatic endocrine cells; HPA: general cytoplasmic expression). Compare that pattern with a no-primary control and adjacent intact tissue under the same DAB conditions (standard IHC validation practice). Treat nuclear-only deposits, staining confined to cut edges or necrosis, and color also present without primary antibody as possible artefacts (UniProt Q9BUX1 subcellular location; standard IHC practice). Endogenous peroxidase can produce misleading chromogen deposition, so verify the peroxidase block when background persists (standard chromogenic IHC practice). Interpret weak or discordant regions cautiously because HPA reports low consistency between antibody staining and RNA expression (HPA: reliability description).
Boster reagents
Best CHAC1 / Glutathione-specific gamma-glutamylcyclotransferase 1 IHC Antibodies
A09017-2 has real IHC data from a paraffin-embedded human pancreas section (IHC image caption); its listed reactivity is Human, Mouse and Rat (catalog). No IF data are supplied (catalog).
Real IHC data
Anti-CHAC1 Antibody ®
Cat # A09017-2
A09017-2 will render with an IHC figure from a paraffin-embedded human pancreas section (IHC image caption). Its listed applications are ELISA, IHC and WB, and its listed reactivity is Human, Mouse and Rat; no IF validation is supplied (catalog).
Which to pick: For tissue IHC, choose A09017-2: it is a polyclonal antibody listed for IHC (catalog), with a paraffin-section image using EDTA retrieval at pH 8.0 (IHC image caption); the fixative is unreported (IHC image caption). For IF/ICC, A09017-2 has no listed application or image data, so its suitability is unverified (catalog). For cross-species IHC, A09017-2 lists Mouse and Rat reactivity, but its supplied IHC figure shows only human pancreas (catalog; IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9BUX1 (CHAC1_HUMAN, Glutathione-specific gamma-glutamylcyclotransferase 1).
- Human Protein Atlas. CHAC1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CHAC1 subcellular location (ICC-IF): Localized to the mitochondria..
- Human Protein Atlas. CHAC1 antibody validation summary (2 antibodies).
- CHAC1 overexpression in human gastric parietal cells with Helicobacter pylori infection in the secretory canaliculi. Helicobacter 2019 — PMC6618068.
- Ferroptosis-related gene CHAC1 is a valid indicator for the poor prognosis of kidney renal clear cell carcinoma. Journal of cellular and molecular medicine 2021 — PMC8034464.
- Helicobacter pylori induces somatic mutations in TP53 via overexpression of CHAC1 in infected gastric epithelial cells. FEBS open bio 2018 — PMC5881537.
- CHAC1 blockade suppresses progression of lung adenocarcinoma by interfering with glucose metabolism via hijacking PKM2 nuclear translocation. Cell death & disease 2024 — PMC11455913.
- PubMed PMID:16572171 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:19109178 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CHAC1 (Glutathione-specific gamma-glutamylcyclotransferase 1). Expected localisation General cytoplasmic staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A09017-2). Validated IHC antibody: A09017-2 at 1:50 recommended; image 2 μg/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 human pancreas tissue; fixative not specified (datasheet A09017-2); 2. Fixation: Image fixative and duration unreported (datasheet A09017-2); 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 A09017-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A09017-2); 8. Primary antibody: Rabbit anti-CHAC1, 1:50 recommended; image 2 μg/ml (datasheet A09017-2); 9. Primary incubation: Overnight at 4 °C (datasheet A09017-2). Watch for: Fixation: Keep fixation consistent (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 A09017-2); Caveat: Staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope overlap is unknown (UniProt); Regulation: UPR-linked; intensity effect unproven (UniProt). Evidence: UniProt Q9BUX1, HPA tissue IHC · Approved, HPA ICC-IF · mitochondria., Datasheet A09017-2, PMC6618068, PMC8034464. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CHAC1/immunohistochemistry-ihc-guide.