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CKMT2 / Creatine kinase S-type, mitochondrial · IHC design guide
Design Immunohistochemistry for CKMT2
Plan CKMT2 chromogenic IHC around cytoplasmic staining in cardiomyocytes and skeletal myocytes (HPA tissue IHC). Use the IHC-validated antibody at 1:50–1:200 (datasheet), and assess distal-tubule staining carefully (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CKMT2 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 IHC staining (HPA tissue IHC); mitochondrial inner membrane is the molecular location (UniProt) | |
| Staining pattern | Cardiomyocytes: high; skeletal myocytes: medium, cytoplasmic (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Heart muscle+2 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific) | |
| Caveat | High distal-tubule staining warrants specificity controls (HPA tissue IHC) | |
| Regulation | Sarcomere-specific expression (UniProt) | |
| Isoform / epitope | No isoforms listed; the mature chain starts at residue 40 (UniProt) |
Section 1
Recommended CKMT2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published CKMT2 staining methods for colorectal tissues and FFPE sections (PMC11216189; PMC10764887; PMC13006575).
| Sample | Paraffin-embedded human tonsil cancer tissue; fixative not specified (datasheet A11457-1) |
| Fixation | Image fixative and duration unreported (datasheet A11457-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-CKMT2, 1:50-1:200 (datasheet A11457-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CKMT2-positive staining in cardiomyocytes of heart muscle (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression mainly in heart and skeletal muscle. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval); EDTA pH 9.0 is a published FFPE alternative (PMC10764887).
Section 2
What Is the Expected CKMT2 Staining Pattern?
CKMT2 is associated with the mitochondrial inner membrane and has no transmembrane segment (UniProt P17540 topology). In paraffin-section IHC, expect cytoplasmic staining mainly in cardiomyocytes and skeletal myocytes (HPA tissue IHC: heart High; skeletal muscle Medium). HPA also reports High staining in kidney distal tubules, despite UniProt describing CKMT2 as restricted to heart and skeletal muscle (HPA tissue IHC; UniProt P17540 tissue specificity). HPA rates the tissue staining evidence Enhanced but notes medium consistency with RNA (HPA tissue IHC).
| Cardiomyocyte cytoplasm stains strongly; skeletal myocyte cytoplasm stains at a moderate level. | This matches the reported IHC distribution and provides a useful comparison between positive tissues (HPA tissue IHC: cardiomyocytes High; skeletal myocytes Medium). Interpret the chromogenic pattern as cellular cytoplasmic staining; inner-membrane placement comes from UniProt, not from resolving that membrane in routine IHC (UniProt P17540 subcellular location). |
| Distinct nuclear or cell-surface staining dominates while expected cytoplasmic staining is weak. | The compartment disagrees with HPA's cytoplasmic tissue profile and UniProt's mitochondrial location (HPA tissue IHC; UniProt P17540 subcellular location). Treat it as suspect until controls and detection conditions have been checked. A visible chromogenic deposit alone does not establish that CKMT2 occupies the stained compartment. |
| Unexpected cells stain, including adipocytes or appendix glandular cells; kidney distal tubules also stain strongly. | HPA reports adipocytes and appendix glandular cells as Not detected, so their staining warrants a cross-reactivity or background check (HPA tissue IHC). Kidney distal tubules require a separate judgment: their High IHC staining is reported by HPA, although it conflicts with UniProt's stated tissue restriction (HPA tissue IHC; UniProt P17540 tissue specificity). |
| Color spreads across tissue and blank spaces, obscuring individual cell boundaries. | That appearance is diffuse background rather than the cell-resolved cytoplasmic profile used to interpret CKMT2 IHC (HPA tissue IHC: cytoplasmic expression mainly in heart and skeletal muscle). In chromogenic IHC generally, inspect the no-primary control and detection reagents before assigning broad color to antigen-specific staining. |
| A heart-muscle section has no cardiomyocyte signal. | A fully negative result conflicts with HPA's High cardiomyocyte staining (HPA tissue IHC). First establish whether the run worked by reviewing tissue preservation, control-slide development, primary-antibody application and detection. The result alone cannot distinguish an assay failure from a sample or antibody problem. |
💡Expected CKMT2 appearanceCall a section positive when cytoplasmic staining is clear in cardiomyocytes and, typically, moderate in skeletal myocytes (HPA tissue IHC: High and Medium); dominant nuclear staining or widespread staining of HPA-negative cells is suspect (HPA tissue IHC; UniProt P17540 subcellular location).
| Localization and topology | CKMT2 is assigned to the mitochondrial inner membrane but has no transmembrane segment (UniProt P17540 topology). Use that assignment to assess compartment plausibility; routine chromogenic IHC need only show a credible cytoplasmic cell pattern (HPA tissue IHC). |
| Tissue distribution and evidence limits | HPA reports High cardiomyocyte, Medium skeletal-myocyte and High kidney distal-tubule staining (HPA tissue IHC). Its Enhanced rating coexists with medium RNA agreement and disregarded presumed off-target staining; kidney staining also differs from UniProt's heart-and-skeletal-muscle restriction (HPA tissue IHC; UniProt P17540 tissue specificity). |
| Processing and isoforms | UniProt lists the mature chain as residues 40–419 and lists no isoforms (UniProt P17540 processing; isoforms). Those facts do not identify the antibody epitope or predict which antigen-retrieval condition will work. Select retrieval from the antibody's IHC instructions and evaluate it with controls (general IHC practice). |
| IF/ICC evidence | HPA provides no CKMT2 ICC/IF images or assigned ICC/IF main location, and the listed antibody has no ICC validation entry (HPA subcellular; HPA antibodies: HPA051880). UniProt's mitochondrial assignment supplies a localization hypothesis, but an IF/ICC result needs validation on its own terms (UniProt P17540 subcellular location). |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in the heart-muscle positive control. | The run may have failed at primary incubation, antigen retrieval or chromogenic detection; HPA reports High cardiomyocyte staining (HPA tissue IHC). | Confirm that the positive-control section developed, check reagent application and follow the IHC-validated antibody's retrieval instructions. Reassess with the same tissue before interpreting negative study sections (general IHC practice). |
| Nuclei or cell outlines stain more strongly than cytoplasm. | The dominant compartment conflicts with the reported cytoplasmic IHC profile and mitochondrial assignment (HPA tissue IHC; UniProt P17540 subcellular location). | Compare a positive tissue and a no-primary control, then review counterstain and detection specificity. Score CKMT2 only where a credible cellular cytoplasmic signal remains (general IHC practice; HPA tissue IHC). |
| Adipocytes or appendix glands show conspicuous color. | Those cell populations are reported as Not detected; antibody cross-reactivity or nonspecific detection is possible (HPA tissue IHC). | Compare staining with no-primary and known-positive controls, and review blocking and chromogen development. Record unexpected staining separately from the expected CKMT2 pattern (general IHC practice; HPA tissue IHC). |
| Kidney distal tubules are strongly positive. | HPA records High distal-tubule IHC staining, while UniProt says CKMT2 is found only in heart and skeletal muscle (HPA tissue IHC; UniProt P17540 tissue specificity). | Report the tissue and cell type explicitly. Compare with heart and HPA-negative controls before assigning the kidney signal to CKMT2; the supplied sources do not resolve this discrepancy. |
| Diffuse color prevents assessment of individual cells. | General IHC causes include nonspecific detection or overdevelopment; the appearance obscures HPA's cell-resolved cytoplasmic pattern (general IHC practice; HPA tissue IHC). | Inspect the no-primary control, blocking and chromogen development, then repeat under conditions that preserve readable cell boundaries (general IHC practice). |
| Can an IF/ICC image be used to confirm the IHC compartment? | HPA supplies no CKMT2 ICC/IF images or assigned main location, and lists no ICC validation for HPA051880 (HPA subcellular; HPA antibodies). | Treat IF/ICC as a separate validation question. Use UniProt's mitochondrial assignment as a hypothesis and assess any IF/ICC signal with appropriate controls; do not infer IF performance from the tissue IHC rating (UniProt P17540 subcellular location; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing CKMT2 staining
A11457-1 · IHCImmunohistochemistry (IHC) analyzes of CKMT2/sMtCK (W262) pAb in paraffin-embedded human tonsil cancer tissue at 1:50.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000131730-CKMT2/tissue/cerebral+cortex#imid_14748500
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000131730-CKMT2/tissue/colon#imid_14772542
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000131730-CKMT2/tissue/liver#imid_14772370
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000131730-CKMT2/tissue/kidney#imid_14772215
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CKMT2 IHC & IF
🧪Run heart muscle first: cardiomyocytes should stain strongly (HPA: High in cardiomyocytes). Use adipose tissue as a negative, with adipocytes at background (HPA: Not detected in adipocytes); non-cardiomyocyte areas on the heart slide should also remain near background, although their CKMT2 status is not specified by the supplied HPA rows.
Positive control tissue: Heart muscle (Cardiomyocytes, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CKMT2; derive a cell-line control from the positive tissue's cell type (Cardiomyocytes) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only) and host-species- and clonality-matched isotype controls, plus a CKMT2 knockout specimen or validated peptide-block control. For chromogenic staining of heart sections, quench endogenous peroxidase and inspect the controls for residual background (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and its effect on staining are unreported; the selected A11457-1 paraffin-section caption does not state a fixative (selected-SKU tissue-IHC caption). CKMT2-specific retrieval requirements are unreported, so assess antigen retrieval during assay optimisation; the available paraffin IHC caption and absence of HPA ICC-IF images do not establish whether frozen sections or IF would be easier (selected-SKU tissue-IHC caption; HPA subcellular). For heart IF, assess intrinsic tissue autofluorescence with an unstained section (standard IF practice).
HPA tissue IHC evidence for CKMT2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded.). 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 |
|---|---|---|---|---|
| Heart muscle | Cardiomyocytes | High | Protein (IHC) | HPA → |
| Kidney | Distal tubules | High | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CKMT2 IHC Tips
Troubleshoot CKMT2 staining in paraffin sections by checking retrieval, compartment, cell type and controls before interpreting chromogenic signal.
How should I retrieve CKMT2 in paraffin sections when staining is weak?
Start with citrate pH 6.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule). Keep section thickness, heating vessel and cooling time consistent across runs so a change in signal can be traced to retrieval rather than handling (general IHC practice). If staining remains weak, compare a modestly longer retrieval with the original condition on adjacent sections, watching for tissue damage or increased background (general IHC practice). Use heart muscle cardiomyocytes as a positive control and record whether their cytoplasmic signal improves before adopting a revised condition (HPA: High in cardiomyocytes; HPA: cytoplasmic profile).
Could fixation explain weak or uneven CKMT2 staining?
CKMT2-specific sensitivity to fixation is unknown from the supplied evidence, so do not assign weak staining to a particular fixative or fixation duration (selected A11457-1 caption: fixative unreported). The catalog image shows staining in paraffin-embedded human tonsil cancer tissue at 1:50, but its caption does not establish how that tissue was fixed (selected A11457-1 caption). For troubleshooting, compare sections with documented processing histories using the same citrate pH 6.0 retrieval and detection run (page retrieval rule; general IHC practice). Examine morphology and internal staining together, and use a separately processed heart muscle control to distinguish a specimen-level problem from a run-level failure (HPA: High in cardiomyocytes; general IHC practice).
What staining pattern fits CKMT2, and what pattern is suspicious?
Expect a cytoplasmic staining distribution in appropriate cells, potentially granular at light-microscope resolution, because CKMT2 is associated with the mitochondrial inner membrane and the tissue IHC profile is cytoplasmic (UniProt P17540: inner-membrane location; HPA: cytoplasmic profile). Chromogenic IHC cannot by itself resolve which side of that membrane contains the epitope (general IHC practice). Compare cardiomyocytes and skeletal myocytes with neighboring cells on the same section; HPA reports High and Medium staining in those respective cell types (HPA: positive cells). Predominantly nuclear, extracellular or uniformly diffuse staining merits a control review before being scored as CKMT2 (UniProt P17540: mitochondrial location; general IHC practice).
How do processing and epitope location affect CKMT2 IHC interpretation?
The record lists 0 isoforms and a processed CKMT2 chain spanning residues 40–419, but it does not identify this antibody’s epitope (UniProt P17540: isoforms and processing; selected A11457-1 caption). Thus, a change in staining cannot be assigned to loss of a particular terminal sequence without antibody-specific mapping (general IHC interpretation). CKMT2 has no transmembrane segment and includes phosphotyrosine 255 and phosphothreonine 356; neither fact demonstrates that retrieval exposes or masks this antibody’s epitope (UniProt P17540: topology and modified residues). Compare retrieval conditions on matched sections and interpret changes against positive-cell morphology and background rather than assuming an isoform switch (general IHC practice).
How should I investigate CKMT2 by IF alongside this IHC guide?
Treat IF/ICC as a separate assay: the supplied HPA subcellular record has 0 cell lines with ICC/IF images, so it provides no direct IF localisation check (HPA: subcellular record). Multiplex CKMT2 with a marker identifying the expected cell type, such as a cardiomyocyte marker in heart tissue, and verify that the two signals occupy the same cells (HPA: High in cardiomyocytes; general IF practice). Choose a fluorophore channel after examining unstained tissue for autofluorescence, and include single-label controls when signals overlap (general IF practice). Because the antibody epitope and its membrane-facing side are unspecified, titrate permeabilisation on matched samples rather than presuming an inner-membrane-facing epitope is accessible (UniProt P17540: inner-membrane location; general IF practice).
How can I reduce CKMT2 background without losing true staining?
First inspect a no-primary control, since endogenous peroxidase activity or nonspecific detection can produce chromogen independently of the CKMT2 antibody (general chromogenic IHC practice). Apply a peroxidase block before DAB detection, then compare background after blocking and washes under otherwise identical conditions (general chromogenic IHC practice). If background rises with retrieval, return to citrate pH 6.0 at 95–98 °C for 20 min before changing antibody concentration (page retrieval rule; general IHC practice). Keep the catalog image’s 1:50 dilution in context as an image condition, and judge any dilution adjustment against signal in heart cardiomyocytes and a no-primary section (selected A11457-1 caption; HPA: High in cardiomyocytes).
What should I measure when comparing CKMT2 IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the eligible cell population before scoring: cardiomyocytes and skeletal myocytes are supported positive populations, whereas mixed tissue can change the apparent overall signal through differences in cell composition (HPA: positive cells; general IHC practice). Use an H-score or report the percentage of positive eligible cells with intensity categories, keeping thresholds and DAB development consistent across slides (general chromogenic IHC practice). Normalise to the number of eligible cells or their sampled tissue area rather than total section area, and record excluded necrotic or damaged regions (general IHC practice). Report matched heart muscle controls alongside each batch so a shift in staining strength can be separated from a shift in specimen composition (HPA: High in cardiomyocytes; general IHC practice).
When does CKMT2 staining warrant caution despite a strong DAB signal?
Give greatest weight to cytoplasmic staining in morphologically intact cardiomyocytes or skeletal myocytes, consistent with CKMT2’s mitochondrial location and the HPA tissue profile (UniProt P17540: inner-membrane location; HPA: cytoplasmic profile and positive cells). Strong nuclear or extracellular signal, section-edge staining, necrotic areas or signal retained in a no-primary control should trigger review for artefact or endogenous enzyme activity (general chromogenic IHC practice). Do not dismiss kidney distal-tubule staining automatically: HPA reports it as High, despite the narrower heart-and-skeletal-muscle tissue specificity in UniProt (HPA: High in distal tubules; UniProt P17540: tissue specificity). Resolve such discrepancies with cell-level morphology, matched controls and independent evidence before calling a CKMT2-positive population (general IHC practice).
Boster reagents
Best CKMT2 / Creatine kinase S-type, mitochondrial IHC Antibodies
A11457-1 is listed for CKMT2 IHC and IF in human, mouse, and rat samples (catalog: applications/reactivity). Its IHC figure shows paraffin-embedded human tonsil cancer tissue (IHC image caption).
Real IHC data
Anti-CKMT2 (W262) Antibody
Cat # A11457-1
A11457-1 will render with an IHC figure of paraffin-embedded human tonsil cancer tissue at 1:50 (IHC image caption). It is listed for IHC and IF in human, mouse, and rat, but has no supplied IF figure (catalog: applications/reactivity/IF image alts).
Which to pick: For tissue IHC, choose A11457-1: its own figure documents paraffin-embedded human tonsil cancer tissue at 1:50; the fixative is unreported (A11457-1 IHC image caption). For IF, A11457-1 is listed as applicable, while ICC validation is unreported (catalog: applications). For cross-species studies, A11457-1 lists human, mouse, and rat reactivity and is rabbit polyclonal; its supplied IHC figure shows human tissue only (catalog: reactivity/host/dilution_raw; IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P17540 (KCRS_HUMAN, Creatine kinase S-type, mitochondrial).
- Human Protein Atlas. CKMT2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CKMT2 subcellular location (ICC-IF): Highest expression in SK-MEL-30: 2.9 nTPM.
- Human Protein Atlas. CKMT2 antibody validation summary (1 antibodies).
- Creatine kinase mitochondrial 2 promotes the growth and progression of colorectal cancer via enhancing Warburg effect through lactate dehydrogenase B. PeerJ 2024 — PMC11216189.
- Heterogeneity-based stratification identifies CKMT2 as a prognostic marker in osteosarcoma. Frontiers in cell and developmental biology 2026 — PMC13247562.
- Prognostic value of mitochondrial CKMT2 in Pan-cancer and its tumor immune correlation analysis. Scientific reports 2024 — PMC10764887.
- Metabolic profiling of the TME uncovers the contrasting impacts of CKMT2 and PDE2A in CRC progression and therapeutic response. Frontiers in pharmacology 2026 — PMC13006575.
- PubMed PMID:2324105 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:2914937 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CKMT2 (Creatine kinase S-type, mitochondrial). Expected localisation Cytoplasmic IHC staining (HPA tissue IHC); mitochondrial inner membrane is the molecular location (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A11457-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Heart muscle (Cardiomyocytes); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil cancer tissue; fixative not specified (datasheet A11457-1); 2. Fixation: Image fixative and duration unreported (datasheet A11457-1); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-CKMT2, 1:50-1:200 (datasheet A11457-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: High distal-tubule staining warrants specificity controls (HPA tissue IHC); Isoform / epitope: No isoforms listed; the mature chain starts at residue 40 (UniProt); Regulation: Sarcomere-specific expression (UniProt). Evidence: UniProt P17540, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in SK-MEL-30: 2.9 nTPM, Datasheet A11457-1, PMC11216189, PMC10764887. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CKMT2/immunohistochemistry-ihc-guide.