CPT1A Antibody

What is CPT1A and why is it significant in research?

CPT1A is a key enzyme in fatty acid oxidation (FAO) that catalyzes the rate-limiting step in this process. Located in the outer mitochondrial membrane, it plays a crucial role in energy metabolism by facilitating the transport of long-chain fatty acids into mitochondria. CPT1A has gained significant research interest because of its abnormal expression in various diseases, particularly cancers. It has been implicated in cancer cell survival, proliferation, and drug resistance, making it an appealing druggable target for cancer therapies . Additionally, CPT1A mutations have been associated with metabolic disorders, highlighting its importance in normal physiological function.

Which cell lines show reliable CPT1A expression for positive controls?

Based on validated experimental data, several cell lines consistently express detectable levels of CPT1A and are suitable as positive controls:

When establishing a new CPT1A antibody-based assay, these cell lines provide reliable positive controls for various applications including Western Blot (WB), Immunoprecipitation (IP), Immunofluorescence/Immunocytochemistry (IF/ICC), and Flow Cytometry (FC) .

What are the optimal dilutions for different applications of CPT1A antibody?

The dilution of CPT1A antibody varies significantly depending on the specific application. Based on validated experimental data, the following dilutions are recommended:

It's important to note that these are general recommendations, and optimal dilutions may vary between specific antibody lots and experimental conditions. Therefore, it is advisable to titrate the antibody in each testing system to obtain optimal results .

How can I optimize antigen retrieval for CPT1A IHC in different tissue types?

Antigen retrieval is a critical step for successful immunohistochemical detection of CPT1A. For optimal results with CPT1A antibody in IHC applications, the following protocol is recommended:

• Primary antigen retrieval should be performed with TE buffer at pH 9.0

• As an alternative approach, citrate buffer at pH 6.0 can also be effective

• For tissues with high fat content (like liver): Extend the antigen retrieval time by 2-3 minutes

• For fibrous tissues: Include a protease digestion step before antigen retrieval

• For each new tissue type: Perform a dilution series experiment with both retrieval buffers to determine optimal conditions

Remember that over-retrieval can lead to nonspecific staining while under-retrieval results in weak signal. Document the optimization process methodically for reproducibility.

What is the most sensitive method for detecting CPT1A activity in research samples?

Traditional methods for measuring CPT1A activity have relied heavily on tritium-labeled L-[³H]carnitine, which offers exceptional sensitivity but presents challenges related to operational safety and scalability due to radioactive waste handling requirements .

A more practical and scalable approach involves a colorimetric method based on CoA detection through thiol-disulfide exchange. This method:

• Detects free thiols from the release of CoA from palmitoyl-CoA during CPT1 catalysis

• Employs 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), which reacts with free CoA to produce 2-thio-5-nitrobenzoic acid (TNB)

• Generates a measurable optical readout at 412 nm

This assay has been validated with known CPT1 inhibitors and can be easily adapted to a 96-well format for high-throughput screening. The method doesn't require purified recombinant CPT1A, making it more accessible for most research laboratories .

How can I generate catalytically active CPT1A for in vitro experiments?

Obtaining catalytically active CPT1A presents a significant challenge as commercially available recombinant CPT1A is typically catalytically inactive due to its requirement for a mitochondrial membrane environment for proper structure and function.

A validated approach involves direct expression of CPT1A in Expi293 cells followed by isolation of mitochondrial extracts:

• Transfect Expi293 cells with a CPT1A expression plasmid

• Isolate mitochondrial extracts from the transfected cells

• Confirm CPT1A expression using ELISA with the following protocol:

  • Coat 100 μg of total protein from cell pellet and supernatant in coating buffer (NaHCO₃-Na₂CO₃, pH 6.4)

  • Block with 2.5% non-fat dry milk in PBST

  • Incubate with mouse anti-human CPT1A monoclonal antibody (1:1,000 dilution)

  • Detect with goat-anti-mouse HRP secondary antibody

This method provides a reliable and scalable source of catalytically active human CPT1A for various experimental applications .

Why might I observe discrepancies between CPT1A protein levels and activity in my samples?

Discrepancies between CPT1A protein levels (detected by antibody-based methods) and enzymatic activity are common and can result from several factors:

• Post-translational modifications: CPT1A activity can be regulated by phosphorylation, acetylation, or other modifications that don't affect antibody detection but alter activity.

• Inhibitory metabolites: Malonyl-CoA is a physiological inhibitor of CPT1A. Varying levels of this metabolite in samples can cause different activity levels despite similar protein expression.

• Membrane environment integrity: CPT1A requires the mitochondrial membrane environment for proper conformation and activity. Sample preparation methods that disrupt this environment may preserve immunogenicity but compromise activity.

• Isoform-specific detection: Some antibodies may detect multiple CPT1 isoforms or cross-react with CPT2, leading to discrepancies when comparing to isoform-specific activity assays.

To troubleshoot, consider performing:

• Western blot with antibodies targeting specific post-translational modifications

• Activity assays with and without malonyl-CoA to assess inhibition sensitivity

• Careful isolation of mitochondrial fractions with membrane integrity preservation

• Validation with isoform-specific knockdown controls

What are common false positives in CPT1A immunostaining and how can they be eliminated?

• Cross-reactivity with other CPT isoforms: CPT1A antibodies may detect the closely related CPT1B, CPT1C, or CPT2 proteins.

  • Solution: Use knockout/knockdown controls or tissues known to express only specific isoforms

  • Validate with multiple antibodies targeting different epitopes

• Non-specific binding in lipid-rich tissues: CPT1A's association with lipid metabolism means it's often expressed in lipid-rich tissues, which can also show non-specific antibody binding.

  • Solution: Use more stringent blocking protocols with BSA and non-fat milk combinations

  • Include additional washing steps with higher detergent concentrations

• Endogenous peroxidase activity: Particularly in liver tissues where CPT1A is highly expressed.

  • Solution: Include a peroxidase quenching step (3% H₂O₂ for 10 minutes) before antibody incubation

• Nuclear false positives: While CPT1A is mitochondrial, non-specific nuclear staining can occur.

  • Solution: Co-stain with mitochondrial markers to confirm localization

  • Use confocal microscopy to verify subcellular localization

Always include appropriate negative controls: isotype controls, secondary antibody-only controls, and when possible, tissues from CPT1A knockout models or cells with CRISPR-mediated CPT1A deletion.

How can CPT1A antibodies be used to investigate the relationship between fatty acid oxidation and cancer progression?

CPT1A antibodies provide valuable tools for investigating the complex relationship between fatty acid oxidation (FAO) and cancer progression. Advanced research applications include:

• Tumor tissue microarray analysis:

  • Use CPT1A antibodies (1:500-1:2000 dilution) for IHC on tumor microarrays to correlate expression levels with clinical outcomes

  • Compare expression between primary tumors and metastatic sites to assess changes during disease progression

  • Combine with markers of lipid metabolism, hypoxia, and proliferation for comprehensive pathway analysis

• Metabolic reprogramming studies:

  • Track changes in CPT1A expression during metabolic adaptation to therapeutic pressure

  • Investigate CPT1A-mediated resistance mechanisms by comparing expression in treatment-naive versus resistant cells

  • Research has shown that CPT1A overexpression indicates poor clinical prognosis in acute myeloid leukemia, highlighting its potential as a biomarker for treatment resistance

• Metastasis and invasion mechanism research:

  • CPT1A has been reported to promote anoikis-resistance and metastasis in colorectal cancer

  • Use CPT1A antibodies in combination with invasion assays to correlate expression with metastatic potential

  • Perform co-immunoprecipitation studies to identify novel CPT1A-interacting proteins in the metastatic cascade

• Combination therapy development:

  • Use CPT1A antibodies to monitor target engagement of CPT1A inhibitors

  • Investigate synergistic effects, such as those observed when combining the CPT1A-selective inhibitor ST1326 with the Bcl-2 inhibitor ABT199 in AML treatment

These applications require rigorous validation of antibody specificity and careful control of experimental conditions to generate reliable and reproducible results.

What are the technical considerations when using CPT1A antibodies to study its role in multiple sclerosis models?

Investigating CPT1A in multiple sclerosis (MS) models presents unique technical challenges that researchers should address:

• Selection of appropriate MS models:

  • Experimental autoimmune encephalomyelitis (EAE) models are commonly used to study MS mechanisms

  • CPT1A mutations, such as the P479L variant, have been shown to affect MBP expression in EAE mice

  • Consider both wild-type and CPT1A variant models for comparative studies

• Brain region-specific analysis:

  • CPT1A expression varies across brain regions and cell types

  • Use precise microdissection techniques to isolate specific regions

  • Perform cell-type specific analysis through co-staining with neuronal, oligodendrocyte, and glial markers

• Protocol adaptations for neural tissue:

  • For Western blot: Use specialized extraction buffers containing 1% Triton X-100 and 0.1% SDS to effectively solubilize membrane-bound CPT1A

  • For IHC/IF: Extended fixation times (24-48h) with 4% PFA are recommended for myelin-rich tissues

  • For optimal results in myelin-rich areas, antigen retrieval should be performed with TE buffer at pH 9.0

• Myelin protein quantification correlation:

  • In EAE models, MBP isoforms (14.0, 17.0, 18.5, and 21.5 kDa) should be measured alongside CPT1A

  • Research has shown that CPT1A mutations can significantly affect MBP expression, with MBP isoforms in CPT1A P479L EAE mice expressed at nearly three-fold higher levels than in wild-type EAE mice

  • Standardize loading controls carefully, as housekeeping protein expression may vary in demyelinating conditions

These technical considerations help ensure that CPT1A antibody-based experiments in MS models generate reproducible and physiologically relevant results.

How can I develop a high-throughput screening assay for CPT1A inhibitors using antibody-based detection methods?

Developing a high-throughput screening (HTS) assay for CPT1A inhibitors requires careful consideration of both enzymatic activity and antibody-based detection methods:

• Enzyme source optimization:

  • Direct expression of CPT1A in Expi293 cells followed by mitochondrial extraction provides a reliable source of catalytically active enzyme

  • Validate CPT1A expression in the extracts using ELISA with anti-CPT1A antibodies prior to screening

  • For reproducibility, prepare large batches of mitochondrial extracts and store at -80°C in single-use aliquots

• Primary activity-based screening:

  • Adapt the colorimetric DTNB-based CoA detection method to a 96-well format

  • Optimize reaction components:

    • CPT1A-containing mitochondrial extracts (standardized by protein content)

    • Palmitoyl-CoA (typically 50-200 μM)

    • L-carnitine (typically 1-5 mM)

    • DTNB for CoA detection (typically 0.1-0.3 mM)

  • Include known inhibitors (e.g., etomoxir, perhexiline) as positive controls

  • Z' factor should exceed 0.5 for a robust HTS assay

• Secondary antibody-based validation:

  • After identifying hits, confirm CPT1A binding using:

    • Antibody-based thermal shift assays to detect compound-induced stability changes

    • Competitive binding assays using labeled antibodies and flow cytometry

    • ELISA-based displacement assays with CPT1A-specific antibodies

• Advanced characterization of confirmed hits:

  • Determine selectivity against other CPT isoforms using isoform-specific antibodies

  • Assess cell permeability and target engagement in intact cells using cellular thermal shift assays with CPT1A antibody detection

  • Evaluate effects on mitochondrial morphology and function through high-content imaging with CPT1A and mitochondrial marker antibodies

This integrated approach combines the throughput of enzymatic screening with the specificity of antibody-based validation methods to identify and characterize novel CPT1A inhibitors effectively.

How might CPT1A antibodies be utilized in developing personalized medicine approaches for metabolic diseases?

CPT1A antibodies can play a crucial role in developing personalized medicine approaches for metabolic diseases through several innovative applications:

• Patient stratification biomarkers:

  • CPT1A expression levels determined through immunohistochemistry may predict response to metabolism-targeting therapies

  • Differential expression patterns could identify patient subgroups most likely to benefit from specific treatments

  • Antibody-based tissue microarray analyses could correlate CPT1A levels with treatment outcomes

• Monitoring therapeutic response:

  • Serial biopsies analyzed with CPT1A antibodies can track changes in expression during treatment

  • Circulating tumor cells can be evaluated for CPT1A expression as a liquid biopsy approach

  • Changes in CPT1A localization or post-translational modifications might serve as early indicators of treatment efficacy

• Detection of functionally relevant CPT1A variants:

  • Develop epitope-specific antibodies that distinguish between key CPT1A variants

  • Mutations in CPT1A have been linked to various metabolic disorders and differential disease progression

  • Antibody-based detection of specific variants could guide treatment selection

• Theranostic applications:

  • CPT1A antibodies conjugated to imaging agents could visualize metabolic activity in vivo

  • Antibody-drug conjugates targeting CPT1A-overexpressing cells represent a potential precision medicine approach

  • Companion diagnostic development using standardized CPT1A immunoassays could identify patients for specific metabolic-targeting therapies

The development of these applications requires rigorous antibody validation, standardized protocols, and correlation with clinical outcomes to translate into effective personalized medicine approaches.

What are the emerging techniques for studying CPT1A protein-protein interactions using antibody-based methods?

Emerging techniques for studying CPT1A protein-protein interactions (PPIs) using antibody-based methods include:

• Proximity ligation assays (PLA):

  • Uses pairs of antibodies against CPT1A and potential interacting partners

  • When proteins are in close proximity (<40 nm), attached oligonucleotides enable rolling circle amplification

  • Provides spatial information about interactions within single cells

  • Particularly valuable for studying mitochondrial membrane-associated interactions of CPT1A

• BioID and TurboID proximity labeling:

  • CPT1A is fused to a biotin ligase (BioID2 or TurboID)

  • Proximal proteins are biotinylated and captured with streptavidin

  • Anti-CPT1A antibodies confirm expression and localization of the fusion protein

  • Ideal for capturing transient or weak interactions in the native cellular environment

• Advanced co-immunoprecipitation techniques:

  • Crosslinking-assisted co-IP using membrane-permeable crosslinkers to stabilize transient interactions

  • Sequential co-IP with antibodies against different domains of CPT1A to identify domain-specific interactions

  • Quantitative co-IP coupled with mass spectrometry for unbiased interactome analysis

  • Published research has identified interacting partners through co-IP methods

• Super-resolution microscopy with dual antibody labeling:

  • STORM or PALM imaging with CPT1A antibodies and interaction partner antibodies

  • Resolution below 20 nm allows visualization of molecular complexes at the mitochondrial membrane

  • Correlative light and electron microscopy (CLEM) provides ultrastructural context for interactions

  • Dynamic interaction studies through live-cell single-molecule tracking with antibody fragments

These techniques are transforming our understanding of CPT1A's functional interactions beyond its enzymatic role, potentially revealing new therapeutic targets and regulatory mechanisms.