CKMT2 Antibody

What is CKMT2 and what is its biological function?

CKMT2 (Creatine Kinase, Mitochondrial 2) is also known as sarcomeric mitochondrial creatine kinase (S-MtCK). It belongs to the creatine kinase isoenzyme family and reversibly catalyzes the transfer of phosphate between ATP and various phosphogens, primarily creatine phosphate. CKMT2 plays a central role in energy transduction in tissues with large, fluctuating energy demands, such as skeletal muscle, heart, brain and spermatozoa .

At the molecular level, CKMT2 activity is associated with oxidative capacity as it increases the availability of ADP to the respiratory chain complex V, which regulates mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) formation. This establishes mitochondria as a hub of innate immune signaling by connecting with pathways involving Toll-like receptors (TLRs) .

CKMT2 shows tissue-specific expression patterns that are important to consider when designing experiments:

Based on immunohistochemistry data from the Human Protein Atlas:

• Strong expression: Heart and skeletal muscle tissues

• Moderate expression: Normal colon and kidney tissues

• Low/no expression: Normal liver tissue

• Variable expression in cancer tissues: Overexpressed in colon cancer (COAD) and cholangiocarcinoma (CHOL), but not expressed in prostate cancer (PRAD) and breast cancer (BRCA)

This expression pattern correlates with tissues having high energy demands, consistent with CKMT2's role in cellular energetics .

What are the optimal conditions for using CKMT2 antibodies in immunohistochemistry?

For optimal IHC-P results with CKMT2 antibodies, consider the following protocol parameters:

• Antigen retrieval: Heat-mediated in EDTA buffer (pH 9.0) is recommended as the primary method; citrate buffer (pH 6.0) can be used as an alternative

• Antibody dilution: 1:500-1:2000 (optimize for each specific antibody)

• Incubation: Overnight at 4°C for primary antibody

• Detection system: HRP-coupled secondary antibody specific to the host species of primary antibody

• Counterstaining: Standard nuclear counterstain (e.g., hematoxylin)

• Positive control recommendations: Mouse heart tissue, human heart tissue

A complete IHC protocol includes dewaxing paraffin sections with methanol, performing antigen retrieval, blocking for 30 minutes, and incubating with CKMT2 antibody (e.g., Proteintech 13207-1-AP at 1:500 dilution) overnight at 4°C, followed by appropriate secondary antibody incubation at room temperature .

How should I validate CKMT2 antibody specificity in my experimental system?

Validating antibody specificity is crucial for reliable results. Consider these approaches:

• Positive controls: Use tissues known to express CKMT2 (heart and skeletal muscle)

• Negative controls: Use tissues with minimal expression (e.g., liver) or omit primary antibody

• Molecular weight validation: Confirm band size on Western blot (expected size: 41-48 kDa)

• siRNA knockdown: Compare antibody signal in control vs. CKMT2-silenced samples

• Overexpression systems: Use CKMT2-overexpressing samples as positive controls

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

For Western blot analysis, CKMT2 is typically detected at 41-48 kDa in heart and skeletal muscle lysates. Expected bands should be validated against predicted molecular weight (48 kDa) .

What sample preparation techniques are critical for CKMT2 detection?

Sample preparation significantly impacts CKMT2 detection quality:

For tissues:

• Fresh-frozen or properly fixed tissues are required

• Formalin fixation and paraffin embedding with proper antigen retrieval is effective for IHC

• For quantification in IHC, software like ImageJ with IHC Profiler plugins can calculate positive area and staining intensity

For cells:

• For ICC/IF: Methanol fixation has been validated for HeLa cells

• For protein extraction: Standard cell lysis buffers with protease inhibitors are suitable

• For mitochondrial protein analysis: Consider mitochondrial isolation protocols to enrich CKMT2 content

For Western blot:

• 10% SDS-PAGE is recommended for optimal separation

• HEK-293T, HeLa cell lysates, and heart/skeletal muscle tissue lysates serve as good positive controls

How is CKMT2 expression altered across different cancer types?

CKMT2 expression varies significantly across cancer types, with important implications for research focus:

• Overexpressed in 4 cancer types:

  • Cholangiocarcinoma (CHOL)

  • Colon adenocarcinoma (COAD)

  • Kidney chromophobe (KICH)

  • Rectum adenocarcinoma (READ)

• Low expression in 14 cancer types:

  • Bladder carcinoma (BLCA)

  • Breast cancer (BRCA)

  • Cervical squamous cell carcinoma (CESC)

  • Esophageal carcinoma (ESCA)

  • Glioblastoma multiforme (GBM)

  • Head and neck squamous cell carcinoma (HNSC)

  • Kidney renal clear cell carcinoma (KIRC)

  • Kidney renal papillary cell carcinoma (KIRP)

  • Lung adenocarcinoma (LUAD)

  • Lung squamous cell carcinoma (LUSC)

  • Pheochromocytoma and paraganglioma (PCPG)

  • Prostate adenocarcinoma (PRAD)

  • Stomach adenocarcinoma (STAD)

  • Uterine corpus endometrial carcinoma (UCEC)

The protein expression data from immunohistochemistry confirms that CKMT2 is strongly expressed in COAD and CHOL but not expressed in PRAD and BRCA, which is consistent with mRNA expression patterns .

What is the prognostic value of CKMT2 in cancer research?

CKMT2 expression has demonstrated prognostic significance:

• Positive prognosis correlation with overexpression:

  • Lung adenocarcinoma (LUAD)

  • Prostate cancer (PRAD)

• Negative prognosis correlation with overexpression:

  • Hepatocellular carcinoma (suggesting poor prognosis and highly malignant potential)

• Stage-specific expression patterns:

  • Lower expression in stage IV compared to stage I in:

    • Bladder carcinoma (BLCA)

    • Head and neck squamous cell carcinoma (HNSC)

    • Lung adenocarcinoma (LUAD)

    • Thyroid carcinoma (THCA)

  • Higher expression in stage IV compared to stage I in:

    • Kidney renal papillary cell carcinoma (KIRP)

These findings suggest CKMT2 may serve as a prognostic biomarker with cancer type-specific significance, warranting tailored approaches to its use in different cancer research contexts .

How does CKMT2 relate to tumor immunity and immunotherapy response?

CKMT2 has emerging significance in cancer immunology:

• Immune pathway enrichment: CKMT2 overexpression is enriched in adaptive immune system and immunoglobulin regulatory pathways

• Tumor microenvironment correlation: Seven cancer types showed positive correlation between low CKMT2 expression and tumor microenvironment characteristics

• Immunotherapy response: In five cancer types, low CKMT2 expression correlated with better immunotherapy treatment outcomes

• Immune cell infiltration: CKMT2 expression levels correlate with the extent of immune cell infiltration in various cancer types. Pearson correlation analysis can be used to quantify this relationship

• Immune-related gene correlation: Strong correlations exist between CKMT2 and most immune-related genes in specific cancer types

These findings suggest CKMT2 plays an important role in cancer immunity and could serve as a potential target for cancer immunotherapy .

What role does CKMT2 play in mitochondrial function in type 2 diabetes?

CKMT2 appears to be a key mediator of mitochondrial dysfunction in type 2 diabetes:

• Expression changes: CKMT2 protein is significantly reduced in the mitochondrial proteome of patients with type 2 diabetes

• Metabolite alterations: Men with type 2 diabetes show:

  • Increased plasma creatine levels

  • Reduced intramuscular phosphocreatine content

• Functional consequences: Reduced CKMT2 contributes to:

  • Impaired mitochondrial respiration

  • Decreased membrane potential

  • Reduced glucose oxidation

  • Disrupted mitochondrial dynamics

• Mechanistic independence: CKMT2's effects on mitochondrial function appear to be independent of:

  • Insulin action

  • Creatine metabolism and availability

This suggests that the CKMT2 reduction in skeletal muscle from people with type 2 diabetes represents a potential molecular link between lipid overload-mediated impairments of mitochondrial function and metabolism .

How does silencing or overexpression of CKMT2 affect cellular metabolism?

Experimental manipulation of CKMT2 produces specific metabolic effects:

CKMT2 silencing effects:

• Reduced basal and maximal mitochondrial respiration

• Decreased mitochondrial membrane potential

• Increased presence of smaller, fragmented mitochondria

• Reduced basal glucose oxidation

• Increased susceptibility to lipid-induced metabolic stress

CKMT2 overexpression effects:

• Increased mitochondrial respiration (independent of creatine availability)

• Mitigated lipid-induced activation of p38 MAPK (a stress- and ROS-responsive kinase)

• Upregulated mRNA levels of antioxidant genes (SOD2, CAT) in palmitate-exposed myotubes

• Improved oxidative phosphorylation in muscle tissue

• Protected against metabolic stress associated with lipid overload

These findings demonstrate that CKMT2 has functions beyond its canonical role in creatine phosphorylation, playing a critical role in maintaining mitochondrial function and integrity .

How does exercise influence CKMT2 expression and function?

Exercise appears to be a positive regulator of CKMT2:

• Expression regulation: Analysis of public data reveals that CKMT2 content is up-regulated by exercise training in both humans and mice

• Functional implications: Since CKMT2 overexpression improves mitochondrial function, exercise-induced increases in CKMT2 may contribute to:

  • Enhanced mitochondrial respiration

  • Improved oxidative phosphorylation

  • Protection against metabolic stress

  • Better glucose metabolism

• Therapeutic potential: The exercise-CKMT2-mitochondrial function relationship suggests that therapeutic strategies targeting CKMT2 expression may be beneficial for metabolic diseases

These findings provide a mechanistic link between exercise, CKMT2 upregulation, and improved mitochondrial function, which may partially explain the beneficial effects of exercise on metabolic health .

How can I optimize CKMT2 antibody use in multiplex immunofluorescence studies?

For multiplex immunofluorescence incorporating CKMT2:

• Antibody selection: Choose an antibody validated for immunofluorescence applications (e.g., ab151450 has been validated in methanol-fixed HeLa cells)

• Fixation optimization:

  • Methanol fixation has been validated for CKMT2 immunofluorescence

  • Test alternative fixation methods for compatibility with other target proteins

• Antibody pairing considerations:

  • Select antibodies raised in different host species to prevent cross-reactivity

  • For co-localization with mitochondrial markers, consider antibodies against:

    • ANT (adenine nucleotide translocator)

    • VDAC (voltage-dependent anion channel)

    • Other mitochondrial proteins for studying CKMT2's role in mitochondrial membrane contact sites

• Detection system:

  • Use appropriate fluorophore-conjugated secondary antibodies

  • Consider nuclear counterstain (e.g., Hoechst 33342) as validated in published protocols

• Control samples:

  • Include CKMT2-silenced cells as negative controls

  • Use heart or skeletal muscle tissue/cells as positive controls

What are the best experimental models for studying CKMT2 function in disease?

Several experimental models have been validated for CKMT2 research:

Cell models:

• C2C12 myotubes: Validated for siRNA knockdown and overexpression studies of CKMT2

• HEK-293T and HeLa cells: Used for antibody validation and expression studies

Animal models:

• C57BL/6 mice fed high-fat diet: Model for studying CKMT2 in insulin resistance

• Exercise-trained mice: Model for studying exercise effects on CKMT2 expression

Human samples:

• Skeletal muscle biopsies from individuals with and without type 2 diabetes

• Tumor and matched normal tissues for cancer studies

Experimental approaches:

• siRNA knockdown for loss-of-function studies

• Electroporation-mediated overexpression for gain-of-function studies

• Ex vivo respirometry assays for functional assessment

• Creatine or β-GPA (creatine analog) supplementation for metabolic studies

These models allow researchers to investigate CKMT2's role in various disease contexts, including cancer, diabetes, and other metabolic disorders .

How should I interpret discrepancies between CKMT2 mRNA and protein expression data?

When faced with discrepancies between CKMT2 mRNA and protein expression:

• Consider post-transcriptional regulation:

  • CKMT2 may be subject to microRNA regulation

  • Long non-coding RNAs (lncRNAs) may influence CKMT2 expression, as they play roles in cancer development

• Evaluate protein stability factors:

  • CKMT2 is highly susceptible to oxidative damage, which may affect protein levels independent of mRNA expression

  • Lipid overload can downregulate CKMT2 expression at the protein level through oxidative damage mechanisms

• Assess tissue-specific translation efficiency:

  • The correlation between mRNA and protein levels varies across tissues

  • Protein expression should be verified in specific tissues of interest

• Technical considerations:

  • Different antibodies may have variable detection sensitivities

  • mRNA quantification methods have different dynamic ranges than protein quantification methods

  • Confirm findings using multiple methodologies (Western blot, IHC, IF) for protein detection

• Functional validation:

  • When discrepancies exist, focus on functional assays (e.g., mitochondrial respiration, membrane potential)

  • Consider the biological context - in some cases, protein function may be more relevant than absolute expression levels

Understanding these factors can help researchers properly interpret discrepancies and design appropriate follow-up experiments to clarify CKMT2's role in their specific research context.

How can I improve CKMT2 antibody sensitivity in tissues with low expression?

For detecting CKMT2 in tissues with naturally low expression:

• Signal amplification techniques:

  • Use tyramide signal amplification (TSA) systems for IHC/IF

  • Consider polymer-based detection systems for enhanced sensitivity

  • Biotin-streptavidin amplification may improve signal-to-noise ratio

• Sample preparation optimization:

  • Test multiple antigen retrieval methods (EDTA pH 9.0 vs. citrate pH 6.0)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize antibody concentration through careful titration

• Detection system selection:

  • HRP-coupled detection systems offer good sensitivity for IHC

  • Fluorescent detection with signal enhancement may improve sensitivity for IF

• Enrichment strategies:

  • For biochemical assays, consider mitochondrial isolation to concentrate CKMT2

  • For tissues with heterogeneous expression, laser capture microdissection may be beneficial

• Antibody selection:

  • Compare multiple antibodies targeting different CKMT2 epitopes

  • Monoclonal antibodies may offer greater specificity but potentially lower sensitivity than polyclonal antibodies

What are the major pitfalls in CKMT2 functional studies and how can they be avoided?

Common pitfalls and their solutions in CKMT2 functional studies: