CKMT1A Monoclonal Antibody

What is CKMT1A and what is its primary biological function?

CKMT1A is a member of the creatine kinase isoenzyme family responsible for transferring high energy phosphate from mitochondria to the cytosolic carrier, creatine. CKMT1A exists as two isoenzymes - sarcomeric MtCK and ubiquitous MtCK - encoded by separate genes. Unlike cytosolic creatine kinase isoenzymes which exist exclusively as dimers, mitochondrial creatine kinase forms both dimers and octamers, providing functional versatility in different cellular contexts .

The primary function of CKMT1A is maintaining energy homeostasis by facilitating the phosphocreatine shuttle system, which effectively connects mitochondrial ATP production with cytosolic ATP utilization. This creates a critical energy buffer system for cells with high and fluctuating energy demands. Notably, CKMT1A has been implicated in cancer metabolism, with numerous malignancies showing overexpression linked to high energy turnover and resistance to apoptosis .

What are the recommended protocols for CKMT1A detection via Western blot?

For optimal Western blot detection of CKMT1A:

• Sample Preparation:

  • Use RIPA buffer for total protein extraction

  • Include protease inhibitors to prevent degradation

  • Load 20-40 μg protein per lane on 10-12% polyacrylamide gels

• Blocking and Antibody Incubation:

  • Block membranes with 5% nonfat milk for 1 hour

  • For mouse anti-human CKMT1A monoclonal antibody: use 1:1000 dilution

  • For rabbit anti-CKMT1A antibody: use 0.04-0.4 μg/mL

  • Incubate primary antibody at 4°C overnight

• Detection and Controls:

  • Use appropriate HRP-conjugated secondary antibodies

  • Tubulin (1:5000) is recommended as an endogenous control

  • Include positive controls (tissues known to express CKMT1A) and negative controls (CKMT1A-knockdown samples)

• Troubleshooting:

  • If background is high, increase blocking time or try different blocking agents

  • If signal is weak, extend incubation time or adjust antibody concentration

  • Prevent antibody freeze-thaw cycles to maintain efficacy

What considerations are important for immunohistochemical detection of CKMT1A?

For successful immunohistochemical detection of CKMT1A in tissue samples:

• Antibody Selection and Dilution:

  • For rabbit anti-CKMT1A antibody: use 1:500-1:1000 dilution

  • Select antibodies validated specifically for IHC applications

• Antigen Retrieval:

  • Heat-induced epitope retrieval is typically required

  • Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) are commonly effective

• Controls and Validation:

  • Include positive control tissues (with known CKMT1A expression)

  • Include negative controls (omitting primary antibody)

  • Verify specificity through peptide competition assays

• Interpretation Guidelines:

  • CKMT1A typically shows cytoplasmic staining with mitochondrial localization

  • In cancer tissues, staining intensity often correlates with pathological grade

  • Evaluate both staining intensity and percentage of positive cells

How can researchers accurately quantify CKMT1A protein levels in biological samples?

Several methodological approaches can be used to quantify CKMT1A levels:

• ELISA:

  • Commercial ELISA kits for human CKMT1A provide sensitive quantification

  • Detection range: 1.57-100 ng/mL

  • Sensitivity: approximately 0.51 ng/mL

  • Suitable for serum, plasma, tissue homogenates, and other biological fluids

  Standard curve reference data:

  Concentration (ng/mL)	OD	Corrected OD
  100.00	2.075	1.967
  50.00	1.502	1.394
  25.00	1.128	1.020
  12.50	0.928	0.820
  6.25	0.539	0.431
  3.13	0.387	0.279
  1.57	0.226	0.118
  0.00	0.108	0.000

• Western Blot Quantification:

  • Use densitometry with appropriate software

  • Always normalize to loading controls (Tubulin recommended)

  • Include standard curves with recombinant CKMT1A protein

  • Consider the dynamic range limitations of Western blotting

• qRT-PCR for mRNA Expression:

  • Design primers specific to CKMT1A

  • Consider potential discrepancies between mRNA and protein levels

  • Use multiple reference genes for accurate normalization

What are the optimal storage conditions for maintaining CKMT1A antibody activity?

To maintain optimal CKMT1A antibody activity:

• Short-term Storage (up to 1 month):

  • Store at 4°C

  • Keep in original container

  • Avoid contamination

• Long-term Storage:

  • Store at -20°C

  • Aliquot to prevent multiple freeze-thaw cycles

  • Expected shelf life: approximately 12 months at -20°C

• Working Solution Handling:

  • Prepare fresh dilutions from stock for each experiment

  • Return stock solution to recommended storage temperature promptly

  • Monitor solution clarity before use (precipitation indicates degradation)

• Critical Precautions:

  • Prevent freeze-thaw cycles which significantly reduce antibody activity

  • Store in formulation containing stabilizers (typical formulation: 1mg/ml in PBS, pH-7.4, with 10% Glycerol and 0.02% Sodium Azide)

  • Document lot numbers and receipt dates for traceability

How does hypoxia regulate CKMT1A expression in cancer cells and what are the implications for cancer progression?

Hypoxia significantly impacts CKMT1A expression through HIF-1α-dependent mechanisms:

• Molecular Mechanism:

  • Under hypoxic conditions (1% O₂), HIF-1α protein accumulates in cancer cells

  • HIF-1α functions as a transcription factor directly regulating CKMT1A expression

  • Western blot analysis demonstrates that hypoxia induces both HIF-1α accumulation and CKMT1A upregulation

• Temporal Expression Pattern:

  • CKMT1A protein levels increase progressively with hypoxia duration

  • Peak expression typically occurs at 24 hours of hypoxia exposure

  • Expression decreases with chronic hypoxia (>24 hours), possibly due to declining cell viability

• Functional Significance:

  • Hypoxia-induced CKMT1A serves as an adaptive mechanism promoting cancer cell survival

  • In non-small cell lung cancer, CKMT1A overexpression correlates with high pathological grade

  • Knockdown of CKMT1A inhibits cell proliferation and invasion, which can be partially rescued by hypoxia

• Experimental Validation:

  • HIF-1 specific inhibitor (LW6) blocks hypoxia-induced CKMT1A upregulation

  • This confirms the HIF-1-dependent regulation mechanism

These findings suggest CKMT1A represents a potential target for cancer hypoxic targeted therapy, particularly in solid tumors where hypoxic microenvironments are common.

What is the optimal protocol for CKMT1A knockdown in cancer cell lines?

For effective CKMT1A knockdown in cancer cell models:

• siRNA Transfection Protocol:

  • Culture cells to 70% confluence in 35-mm dishes

  • Replace with serum-free, antibiotic-free medium 2 hours before transfection

  • Dilute 5 μg siRNA in 250 μl serum-free medium

  • Separately dilute 5 μl Lipofectamine 2000 in 250 μl serum-free medium

  • Mix diluted siRNA and Lipofectamine, incubate at room temperature for 20 minutes

  • Add mixture to cells and incubate at 37°C for 6 hours

  • Replace with complete medium and conduct subsequent experiments after 48-72 hours

• siRNA Sequence Design:

  • Published effective sequence: 5′-ACGGTACCATGGCTGGTCCCTTCTCCCGT-3′

  • Design multiple siRNAs targeting different regions of CKMT1A mRNA

  • Include non-targeting siRNA sequences as negative controls

• Validation of Knockdown:

  • Western blot analysis to confirm protein reduction

  • qRT-PCR to verify mRNA downregulation

  • Functional assays to assess phenotypic consequences

• Alternative Approaches:

  • For stable knockdown, consider lentiviral shRNA delivery systems

  • CRISPR-Cas9 gene editing for complete knockout studies

  • Rescue experiments with siRNA-resistant CKMT1A constructs to confirm specificity

What is the relationship between CKMT1A expression and epithelial-mesenchymal transition (EMT) in cancer progression?

CKMT1A plays a significant role in epithelial-mesenchymal transition (EMT), a critical process in cancer progression:

• Experimental Evidence:

  • Knockdown of CKMT1A inhibits EMT in non-small cell lung cancer cells (H1650 and H1299)

  • Western blot analysis reveals changes in EMT markers following CKMT1A manipulation

  • Hypoxia promotes EMT, which can be partially reversed by CKMT1A knockdown

• EMT Marker Regulation:

  • CKMT1A knockdown leads to:

    • Increased E-cadherin expression (epithelial marker)

    • Decreased N-cadherin expression (mesenchymal marker)

    • Reduced Snail1 expression (EMT transcription factor)

  • These changes indicate a shift toward epithelial phenotype and reduced invasive capacity

• Mechanistic Integration:

  • CKMT1A likely influences EMT through altered energy metabolism affecting cellular plasticity

  • Hypoxia-induced CKMT1A expression may serve as a critical link between the hypoxic microenvironment and EMT

  • The energy shuttle function of CKMT1A may support the high energy demands of invasive cancer cells

• Methodological Approaches:

  • Monitor canonical EMT markers (E-cadherin, N-cadherin, Snail1) after CKMT1A manipulation

  • Assess morphological changes and invasive properties using Transwell assays

  • Combine with hypoxia treatment to model physiological tumor conditions

This relationship provides a mechanistic explanation for how metabolic adaptations and energy metabolism can directly influence cancer cell plasticity and metastatic potential.

How do CKMT1A expression patterns differ across cancer types, and what are the implications for targeted therapy?

CKMT1A exhibits distinct expression patterns and functional roles across cancer types:

• Non-small Cell Lung Cancer (NSCLC):

  • Significantly upregulated compared to normal lung tissue

  • Expression correlates with pathological grade

  • Knockdown inhibits cell proliferation and invasion

  • Associated with hypoxia adaptation via HIF-1α regulation

• Acute Myeloid Leukemia (AML):

  • Particularly relevant in EVI1-positive AML

  • Inhibition reduces viability of AML cell lines

  • Promotes cell cycle arrest and apoptosis when suppressed

• Oral Squamous Cell Carcinoma (OSCC):

  • Distinct functional pattern

  • Overexpression induces apoptosis

  • Limited effect on invasion capacity

  • Suggests context-dependent functions

• Other Malignancies:

  • Generally overexpressed in aggressive cancers with poor prognosis

  • Associated with high energy turnover

  • Contributes to apoptosis resistance

  • May support metabolic adaptations in tumor progression

Implications for targeted therapy:

• Context-Specific Approaches:

  • CKMT1A targeting strategies must consider cancer-specific functions

  • Combined biomarker approach with hypoxia markers may improve patient selection

  • Tumors with high HIF-1α and CKMT1A may be particularly suitable for targeted intervention

• Therapeutic Strategies:

  • Direct CKMT1A inhibition in hypoxic tumors

  • Combination with hypoxia-activated prodrugs

  • Targeting the HIF-1α/CKMT1A axis rather than CKMT1A alone

  • Consideration of both oligomeric forms (dimers vs. octamers) in drug design

What experimental approaches can distinguish between the dimeric and octameric forms of CKMT1A?

Distinguishing between CKMT1A oligomeric forms requires specialized methodologies:

• Native Gel Electrophoresis:

  • Blue Native PAGE preserves native protein complexes

  • Use mild detergents and avoid reducing agents during sample preparation

  • Expected molecular weights:

    • Dimeric CKMT1A: ~80-90 kDa

    • Octameric CKMT1A: ~340-360 kDa

  • Follow with Western blot using specific CKMT1A antibodies

• Size Exclusion Chromatography:

  • Use columns with appropriate fractionation range (Superose 6 or Sephacryl S-300)

  • Maintain physiological buffers to preserve native structures

  • Collect fractions for subsequent activity assays or Western blot

  • Compare elution profiles with molecular weight standards

• Analytical Ultracentrifugation:

  • Sedimentation velocity experiments differentiate species by sedimentation coefficients

  • Sedimentation equilibrium provides information on molecular mass distributions

  • Both approaches require specialized equipment and expertise

• Functional Differentiation:

  • Membrane binding assays (octamers preferentially associate with mitochondrial membranes)

  • Enzymatic activity assays (different kinetic properties between forms)

  • Differential extraction protocols to separate membrane-bound vs. soluble forms

Understanding the balance between these oligomeric forms is crucial as they serve different functions in cellular metabolism and may have different implications in disease contexts.

How can researchers integrate CKMT1A studies with broader investigations of tumor metabolic reprogramming?

Integrating CKMT1A research within the broader tumor metabolic context requires:

• Multi-omics Approaches:

  • Combine CKMT1A-focused studies with:

    • Transcriptomics to identify co-regulated genes

    • Proteomics to map interaction networks

    • Metabolomics to measure effects on global metabolism

  • Integration strategies should include pathway enrichment analysis and network modeling

• Spatial Metabolic Analysis:

  • Map CKMT1A expression relative to:

    • Hypoxic regions (using pimonidazole or HIF-1α staining)

    • Proliferative zones (Ki-67 positive areas)

    • Vascular structures

  • Use multiplex immunofluorescence or spatial transcriptomics approaches

• Energy Transfer Investigations:

  • Study complete phosphocreatine shuttle components

  • Monitor ATP dynamics using FRET-based sensors

  • Combine with mitochondrial function assays

  • Analyze creatine/phosphocreatine ratios in cellular compartments

• Combinatorial Targeting Strategies:

  • Evaluate CKMT1A inhibition alongside:

    • Glycolysis inhibitors

    • Mitochondrial respiration inhibitors

    • Glutaminolysis inhibitors

  • Measure synergistic effects on cell viability and metabolic adaptation

• Therapeutic Translation:

  • Correlate CKMT1A with patient outcomes across cancer types

  • Develop biomarker strategies combining CKMT1A with hypoxia markers

  • Design targeted approaches for tumors with specific metabolic dependencies

This integrated approach can provide deeper insights into how CKMT1A contributes to the complex metabolic landscape of tumors and identify more effective targeting strategies.