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CKAP5 / Cytoskeleton-associated protein 5 · IHC design guide
Design Immunohistochemistry for CKAP5
Plan CKAP5 staining in paraffin sections around the observed cytoplasmic tissue pattern (HPA tissue IHC). This guide covers consistent fixation, the catalog antibody's 2–5 μg/mL IHC range (datasheet A05324), and mitotic localization when interpreting staining (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CKAP5 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 | Observed tissue: cytoplasm (HPA tissue IHC); molecular: centrosome/spindle (UniProt) | |
| Staining pattern | Widespread cytoplasmic staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05324) | |
| Positive control | Adipose tissue+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 sections (standard IHC practice; not target-specific) | |
| Caveat | Mitotic cells may show spindle enrichment (UniProt) | |
| Regulation | Elevated in hepatomas and colonic tumors (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope coverage is undetermined (UniProt) |
Section 1
Recommended CKAP5 IHC & IF Protocols
The catalog antibody protocol is paired with a published CKAP5 IHC protocol for xenograft tumors (PMC10075965).
| Sample | Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A05324) |
| Fixation | Image fixative and duration unreported (datasheet A05324); 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 A05324); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05324) |
| Primary antibody | Rabbit anti-CKAP5, 2-5μg/ml (datasheet A05324) |
| Primary incubation | Overnight at 4 °C (datasheet A05324) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A05324) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CKAP5-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval (datasheet A05324); the published xenograft protocol used acidic retrieval for 20 min (PMC10075965).
Section 2
What Is the Expected CKAP5 Staining Pattern?
CKAP5 should produce a predominantly cytoplasmic tissue IHC pattern, with prominent staining in HPA-listed cells such as adipocytes, cortical neurons, bronchial ciliated cell bodies and colon endothelial cells (HPA: ubiquitous cytoplasmic expression; High in these cells). Centrosomal and spindle-associated localisation is biologically plausible, although those small structures may be difficult to resolve in chromogenic sections (UniProt Q14008: subcellular localisation). The HPA tissue assessment is Supported, with medium consistency between staining and RNA data (HPA: tissue reliability). CKAP5 has no transmembrane segment (UniProt Q14008: topology).
| Broad cytoplasmic colour in intact cells, with stronger staining in an HPA-listed population. | This fits the reported ubiquitous cytoplasmic profile; examples rated High include adipocytes, cortical neurons and bronchial ciliated cell bodies (HPA: tissue IHC). Judge the signal in cell context: a small pericentrosomal focus can also fit CKAP5 localisation, but chromogenic IHC may not resolve it reliably (UniProt Q14008: centrosome; general IHC practice). |
| Signal is confined to nuclei, cell surfaces or extracellular material, without convincing cytoplasmic staining. | Treat this as a compartment mismatch requiring review, because the tissue profile is cytoplasmic and CKAP5 has no transmembrane segment (HPA: tissue IHC; UniProt Q14008: topology). Apparent nuclear colour alone does not establish CKAP5: the HPA nucleolar observations are uncertain subcellular findings, not a validated tissue IHC pattern (HPA: subcellular localisation). |
| Strong colour appears only in an unexpected cell population, while the HPA-described cells lack convincing signal. | Cross-reactivity or endogenous detection activity is possible; inspect a detection control and compare the staining with intact cell morphology (general IHC practice). HPA reports low tissue specificity and ubiquitous cytoplasmic expression, so an unlisted cell type is not automatically CKAP5-negative (HPA: tissue IHC). |
| Colour spreads across tissue, lumen and surrounding spaces, obscuring cell boundaries. | This is diffuse background rather than a readable cellular pattern (general IHC practice). It cannot support the reported cytoplasmic distribution or a comparison between HPA High cell populations and surrounding cells (HPA: tissue IHC). Reassess the background before scoring CKAP5 expression (general IHC practice). |
| A section containing an HPA-listed High cell population has no convincing cellular colour. | First confirm that the expected cells are present and identifiable; examples include adrenal glandular cells, duodenal glandular cells and cerebral cortical neurons (HPA: High in these cells). An absent signal can reflect a failed staining run or detection problem (general IHC practice). HPA's Supported rating does not guarantee positivity in every individual specimen (HPA: tissue reliability). |
💡Expected CKAP5 appearanceCall a positive result when intact HPA-listed cells show clear cytoplasmic staining, potentially High in the specified populations (HPA: tissue IHC); isolated nuclear, surface-only or acellular colour without matching cytoplasm is suspect (HPA: cytoplasmic profile; general IHC practice).
| Choice of tissue and cell population | HPA rates adipocytes, adrenal and duodenal glandular cells, bronchial ciliated cell bodies, cortical neurons and colon endothelial cells High; endometrial glandular cells are Medium (HPA: tissue IHC). These are useful comparison populations, while the reported ubiquitous profile limits the value of assuming an unlisted cell type is negative (HPA: tissue IHC). |
| Antibody evidence | The two listed rabbit polyclonal antibodies, HPA039377 and HPA040375, each have Supported IHC status (HPA: antibody validation). The overall tissue assessment has medium consistency with RNA data (HPA: tissue reliability). These ratings support comparison with the reported pattern; they do not validate every staining feature in an individual section (general IHC practice). |
| Intracellular distribution | UniProt places CKAP5 at centrosomes, spindle poles, spindle microtubules and kinetochores during relevant cell-cycle stages (UniProt Q14008: subcellular localisation). HPA tissue IHC describes the broader observable pattern as cytoplasmic (HPA: tissue IHC). Score routine chromogenic sections at a resolution the cells permit; do not require a visible mitotic structure in every positive cell (general IHC practice). |
| Isoforms and processing | Three isoforms are listed, and the annotated protein chain spans residues 1–2032, with no signal peptide or propeptide (UniProt Q14008: isoforms and processing). The supplied evidence does not map either IHC antibody to a specific epitope, so it cannot establish which isoforms an antibody detects or predict a staining difference between them (HPA: listed antibody validation; UniProt Q14008: isoforms). |
| What should IF/ICC show? | A centrosomal signal is the supported main subcellular observation; basal body, nucleolar, plasma membrane, spindle and primary-cilium observations are marked uncertain by HPA (HPA: subcellular localisation). UniProt also describes CKAP5 at centrosomes and mitotic spindle structures (UniProt Q14008: subcellular localisation). Interpret those observations within the separate IF/ICC guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in an HPA High comparison population. | The expected cells may be absent from the section, or the staining run may have failed (HPA: tissue IHC; general IHC practice). | Confirm cell identity and section integrity, then check the existing IHC antibody and detection controls before interpreting a biological absence (general IHC practice). |
| Uniform colour covers cells and empty spaces. | Excess background or incomplete removal of detection reagents can obscure cellular staining (general IHC practice). | Review the detection control, blocking and washes; score CKAP5 only after cell boundaries and cytoplasmic colour can be distinguished (general IHC practice). |
| Colour is confined to nuclei or tissue edges. | This distribution conflicts with the reported cytoplasmic tissue profile and may be artefactual (HPA: tissue IHC; general IHC practice). | Compare intact internal cells with the affected regions and inspect a detection control before calling the stain positive (general IHC practice). |
| Only an unexpected cell population stains strongly. | Cross-reactivity or endogenous detection activity is possible, but an unlisted cell type is not proven negative by HPA's ubiquitous profile (HPA: tissue IHC; general IHC practice). | Check morphology and the detection control, then compare the result with HPA-described cells in the same tissue (HPA: tissue IHC; general IHC practice). |
| Cytoplasmic staining is present, but no centrosomal dots are visible. | Small centrosomal structures may be unresolved in chromogenic tissue IHC (UniProt Q14008: centrosome; general IHC practice). | Judge the tissue result against the supported cytoplasmic pattern and identified positive cells; do not require a resolvable centrosome (HPA: tissue IHC; general IHC practice). |
| A suspected mitotic cell shows staining that differs from neighbouring cells. | CKAP5 occupies spindle poles, spindle microtubules and kinetochores during mitosis (UniProt Q14008: subcellular localisation). | Check mitotic morphology and surrounding cytoplasmic staining before treating the difference as artefact; avoid assigning a specific spindle structure that the section cannot resolve (UniProt Q14008: subcellular localisation; general IHC practice). |
Experiment evidence
IHC & IF images showing CKAP5 staining
A05324 · IHCIHC analysis of ch TOG/CKAP5 using anti-ch TOG/CKAP5 antibody (A05324). ch TOG/CKAP5 was detected in a paraffin-embedded section of human li
A05324 · IHCIHC analysis of ch TOG/CKAP5 using anti-ch TOG/CKAP5 antibody (A05324). ch TOG/CKAP5 was detected in a paraffin-embedded section of human pr
A05324 · IFIF analysis of ch TOG/CKAP5 using anti-ch TOG/CKAP5 antibody (A05324). ch TOG/CKAP5 was detected in an immunocytochemical section of A431 ce
M05324 · IHCIHC analysis of Ch TOG/CKAP5 using anti-Ch TOG/CKAP5 antibody (M05324). Ch TOG/CKAP5 was detected in paraffin-embedded section of human live
M05324 · IHCIHC analysis of Ch TOG/CKAP5 using anti-Ch TOG/CKAP5 antibody (M05324). Ch TOG/CKAP5 was detected in paraffin-embedded section of human rect
M05324 · IFIF analysis of Ch TOG/CKAP5 using anti-Ch TOG/CKAP5 antibody (M05324). Ch TOG/CKAP5 was detected in an immunocytochemical section of Hela ce
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000175216-CKAP5/tissue/cerebral+cortex#imid_10442957
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000175216-CKAP5/tissue/colon#imid_10442875
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000175216-CKAP5/tissue/liver#imid_10442884
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000175216-CKAP5/tissue/kidney#imid_10442893
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA040375; green = CKAP5, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA040375; green = CKAP5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CKAP5 IHC & IF
🧪Run cerebral cortex first and look for staining in neuronal cells (HPA: High in cerebral cortex neuronal cells). HPA detects CKAP5 in all 45 scored tissues, so use no-primary and isotype controls for the negative; no cell population on the positive slide is established as CKAP5-negative, and any apparently unstained cells should be treated as observations rather than assumed negatives (HPA: no negative rows).
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: None in HPA: CKAP5 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 CKAP5 in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Centrosome (supported), Basal body (uncertain) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a host- and class-matched rabbit IgG isotype control, and CKAP5 knockout material or immunizing-peptide competition if available (caption: rabbit primary antibody; standard IHC practice). Quench endogenous peroxidase for HRP/DAB, and assess neuronal autofluorescence if examining cortex by IF (caption: HRP/DAB; standard IHC/IF practice).
⚠️Feasibility: The selected A05324 paraffin-section caption uses heat-mediated EDTA retrieval at pH 8.0, but reports no fixative; a target-specific fixation window, fixation effect, and retrieval dependency are unreported (A05324 tissue-IHC caption). IF can help resolve centrosomal staining, while cortical autofluorescence needs assessment; frozen-section performance and whether IF is easier than IHC are unreported (HPA: centrosome supported; standard IF practice).
HPA tissue IHC evidence for CKAP5
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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Ciliated cells (cell body) | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: CKAP5 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced CKAP5 IHC Tips
Troubleshoot CKAP5 staining in paraffin sections by checking retrieval, cell compartment, detection background, and scoring before interpreting chromogenic signal.
Which retrieval conditions should I start with for CKAP5 in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A05324). The documented liver cancer section was stained after that retrieval, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A05324). If staining is weak, vary heating duration on adjacent sections while keeping buffer and antibody conditions fixed, then compare signal with tissue morphology (standard IHC practice). Record the heating and cooling conditions because excessive retrieval can damage sections and make apparent staining difficult to interpret (standard IHC practice).
Could fixation explain weak CKAP5 staining?
The selected paraffin-section caption does not state a fixative, so CKAP5-specific sensitivity to fixation is unknown (datasheet A05324). Do not infer that sensitivity from the protein’s cytoplasmic location or phosphorylation sites (UniProt Q14008; datasheet A05324). Compare sections with documented fixation histories under the same EDTA pH 8.0 retrieval and antibody conditions, and inspect morphology alongside staining (datasheet A05324; standard IHC practice). If only poorly documented material is available, report the fixation uncertainty and avoid interpreting weak chromogen as evidence of low CKAP5 expression (standard IHC practice).
Where should convincing CKAP5 staining appear?
Expect predominantly cytoplasmic staining across tissue cells, with centrosome-associated signal potentially appearing as small puncta (HPA tissue IHC; HPA subcellular: centrosome supported). CKAP5 also localizes to spindle poles, spindle microtubules, and kinetochores during mitosis, so interpret such patterns in the context of recognizable dividing cells (UniProt Q14008). HPA lists basal body and several other additional locations with uncertain support; treat isolated signal in those compartments cautiously (HPA subcellular). Review the DAB pattern at high magnification against the counterstain, and score compartment and cell identity separately from staining intensity (standard IHC practice).
How do isoforms and epitope accessibility affect interpretation?
CKAP5 has 3 annotated isoforms, but the supplied antibody caption does not identify its recognized epitope or establish isoform coverage (UniProt Q14008; datasheet A05324). It is a cytoskeletal protein with no annotated transmembrane segment, signal peptide, or glycosylation sites; those annotations do not establish which epitope survives processing (UniProt Q14008). Five modified residues are listed, including phosphoserines at positions 816, 845, 1469, and 1861; their effects on this antibody’s staining are unknown (UniProt Q14008; datasheet A05324). If samples disagree, compare matched retrieval and detection conditions before assigning the difference to an isoform or modification (standard IHC practice).
How should an IF check complement the chromogenic IHC result?
Use IF as a separate check of whether CKAP5 signal falls in the expected cells and compartments; the selected antibody caption documents paraffin-section IHC, not IF validation (datasheet A05324; UniProt Q14008). Multiplex with a validated marker for the cell type being examined, such as an endothelial cell marker when assessing the reported colon endothelial signal (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and assess tissue autofluorescence before assigning faint puncta to CKAP5 (standard IF practice). Because CKAP5 lacks a transmembrane segment and localizes chiefly inside cells, use controlled permeabilisation to expose intracellular epitopes, then compare matched single-marker controls (UniProt Q14008; standard IF practice).
What should I adjust when DAB background obscures CKAP5?
First distinguish diffuse chromogen from cell-associated cytoplasmic staining and check whether the counterstain still reveals intact tissue architecture (HPA tissue IHC; standard IHC practice). The selected section used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase-conjugated secondary for 30 minutes at 37°C (datasheet A05324). Include a no-primary control, block endogenous peroxidase, and check washing and DAB development time when background rises (standard IHC practice). If background persists, titrate primary concentration around the documented condition while judging specific cytoplasmic signal and background on matched sections (datasheet A05324; standard IHC practice).
How should I score CKAP5 staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, because the tissue profile is broadly cytoplasmic and varies by cell type (HPA tissue IHC). For comparable regions, report an H-score from staining intensity and percentage of positive cells, or report percentage positive when intensity categories are unreliable (standard IHC practice). Normalize counts to the number of evaluable cells; if measuring positive-cell density, use cells per mm² of viable tissue (standard IHC practice). Keep retrieval, DAB development, counterstaining, imaging, and scoring thresholds consistent across samples, and exclude folds, edges, and necrotic areas from the analysis (standard IHC practice).
How can I separate true CKAP5 signal from artefact?
Favor reproducible cytoplasmic cell-associated staining, with centrosomal puncta or mitotic spindle patterns when cell morphology supports them (HPA tissue IHC; HPA subcellular: centrosome supported; UniProt Q14008). A strong nuclear-only or uniform extracellular DAB pattern conflicts with the principal reported locations and warrants review of controls and morphology (HPA tissue IHC; UniProt Q14008; standard IHC practice). Check edge-heavy staining, necrotic regions, and residual endogenous peroxidase against a no-primary control before calling a sample positive (standard IHC practice). HPA rates the tissue-IHC evidence as Supported with medium agreement between staining and RNA, so interpret unexpected cell patterns cautiously (HPA tissue IHC).
Boster reagents
Best CKAP5 / Cytoskeleton-associated protein 5 IHC Antibodies
Two anti-CKAP5 antibodies have IHC images from human tumor and rodent brain paraffin sections, plus IF/ICC images from human cells (catalog IHC and IF captions).
Real IHC data
Anti-ch TOG/CKAP5 Antibody ®
Cat # A05324
Real IHC data
Anti-ch TOG/CKAP5 Antibody ® (monoclonal, 3C13)
Cat # M05324
A05324 has IHC images from human liver and prostate cancer and mouse and rat brain, plus an IF/ICC image from A431 cells (A05324 image captions). M05324 has IHC images from human liver, rectal, and renal cancer and rat brain, plus an IF/ICC image from HeLa cells (M05324 image captions).
Which to pick: For tissue IHC across human, mouse, and rat, choose rabbit antibody A05324, which has paraffin-section images in all three species (A05324 host and IHC captions). Choose mouse monoclonal M05324, clone 3C13, when its human or rat paraffin-section images and mouse-primary detection suit the experiment; mouse reactivity is listed, but no mouse IHC image is provided (M05324 catalog and IHC captions). For IF/ICC, A05324 is shown in A431 cells and M05324 in HeLa cells; the fixative used for either antibody’s paraffin-section IHC images is unreported (catalog IF and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q14008 (CKAP5_HUMAN, Cytoskeleton-associated protein 5).
- Human Protein Atlas. CKAP5 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. CKAP5 subcellular location (ICC-IF): Mainly localized to the centrosome and basal body. In addition localized to the nucleoli, nucleoli rim, plasma membrane, mitotic spindle and primary cilium..
- Human Protein Atlas. CKAP5 antibody validation summary (2 antibodies).
- CKAP5 deficiency induces premature ovarian insufficiency. EBioMedicine 2025 — PMC12032925.
- Therapeutic gene silencing of CKAP5 leads to lethality in genetically unstable cancer cells. Science advances 2023 — PMC10075965.
- PubMed PMID:8536682 — UniProt-cited evidence.
- PubMed PMID:7788527 — UniProt-cited evidence.
- PubMed PMID:16554811 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CKAP5 (Cytoskeleton-associated protein 5). Expected localisation Observed tissue: cytoplasm (HPA tissue IHC); molecular: centrosome/spindle (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05324). Validated IHC antibody: A05324 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); 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 liver cancer tissue; fixative not specified (datasheet A05324); 2. Fixation: Image fixative and duration unreported (datasheet A05324); 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 A05324); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05324); 8. Primary antibody: Rabbit anti-CKAP5, 2-5μg/ml (datasheet A05324); 9. Primary incubation: Overnight at 4 °C (datasheet A05324). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Mitotic cells may show spindle enrichment (UniProt); Isoform / epitope: 3 isoforms; epitope coverage is undetermined (UniProt); Regulation: Elevated in hepatomas and colonic tumors (UniProt). Evidence: UniProt Q14008, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the centrosome and basal body. In addition…, Datasheet A05324, PMC10075965. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CKAP5/immunohistochemistry-ihc-guide.