CPT1C Antibody

What is CPT1C and how does it differ from other CPT1 isoforms?

CPT1C belongs to the carnitine/choline acetyltransferase family and is one of three CPT1 isoforms (CPT1A, CPT1B, and CPT1C). Unlike the other isoforms, CPT1C is primarily expressed in the brain, particularly in neurons. While structurally similar to other CPT1s, it has limited carnitine palmitoyltransferase catalytic activity. Research indicates CPT1C functions as a palmitoyl thioesterase specifically expressed in the endoplasmic reticulum of neurons . It binds to palmitoyl-CoA and malonyl-CoA but exhibits very low or undetectable canonical CPT1 catalytic activity .

How should I select between monoclonal and polyclonal CPT1C antibodies for my research?

The choice depends on your specific application:

Monoclonal antibodies (e.g., 66072-1-Ig from Proteintech):

• Offer high specificity targeting a single epitope

• Show greater consistency between batches

• Particularly useful for quantitative applications

• Example: Mouse IgG2b monoclonal (66072-1-Ig) shows reactivity with human and pig samples

Polyclonal antibodies (e.g., 12969-1-AP from Proteintech):

• Recognize multiple epitopes on the antigen

• Generally provide higher sensitivity but potentially lower specificity

• Better for detection of denatured proteins

• Example: Rabbit IgG polyclonal (12969-1-AP) shows reactivity with human, mouse, and rat samples

For critical experiments, validation with knockout/knockdown controls is recommended, as seen in antibody 415 003 which has been KO validated (PubMed: 37309891) .

What are the best positive controls for validating CPT1C antibodies?

Based on the search results, recommended positive controls include:

When working with novel tissues or cell lines, preliminary validation against these established positive controls is advisable to confirm antibody performance .

What are the optimal conditions for CPT1C antibody use in different applications?

The following table summarizes recommended dilutions for common applications:

It is recommended to titrate these antibodies in each testing system to obtain optimal results. Conditions are sample-dependent and validation data galleries should be consulted for specific applications .

What antigen retrieval methods are recommended for CPT1C detection in fixed tissues?

For optimal CPT1C detection in immunohistochemistry:

• Primary recommendation: Antigen retrieval with TE buffer pH 9.0

• Alternative method: Antigen retrieval with citrate buffer pH 6.0

These recommendations are consistent across multiple antibodies in the search results, suggesting these conditions optimize CPT1C epitope exposure in fixed tissues .

How can CPT1C antibodies be used to study its interaction with AMPA receptors?

CPT1C has been identified as an integral component of native AMPA receptor complexes and modulates their surface expression. To study this interaction:

• Co-immunoprecipitation approach:

  • Immunoprecipitate with anti-GluA1-NT antibody (as demonstrated in PubMed study)

  • Alternatively, precipitate with anti-GFP antibody when using CPT1C-GFP constructs

  • Use protein-A sepharose beads (80-100 μl) for pulling down antibody-protein complexes

  • Wash with lysis buffer three times before elution with sample buffer

• Mutation studies:

  • Create CPT1C constructs with mutations in putative thioesterase catalytic residues using site-directed mutagenesis

  • Common mutations include H470A, S252A, and D474A to produce alanine substitutions

  • These mutants allow investigation of the functional domains involved in AMPAR trafficking

This approach has revealed that CPT1C modulates the trafficking of glutamate receptor AMPAR to plasma membrane through depalmitoylation of GRIA1 and regulation of SACM1L phosphatidylinositol-3-phosphatase activity .

How does CPT1C expression correlate with cancer prognosis?

Multiple studies have established CPT1C as a potential prognostic marker:

These findings suggest CPT1C could serve as a valuable prognostic biomarker in multiple cancer types.

What mechanisms underlie CPT1C-mediated cancer progression?

Several key mechanisms have been identified:

• Enhanced fatty acid oxidation (FAO):

  • CPT1C catalyzes carnitinylation of fatty acids for transport into mitochondria

  • Increased FAO rate provides energy for cancer cell proliferation

  • In CRC cells, enforced CPT1C expression increased FAO rate, while silencing decreased it

• Cell cycle regulation:

  • CPT1C overexpression prolongs S phase of the cell cycle

  • Increases expression of proliferating cell nuclear antigen (PCNA) and cyclin D1

  • In gastric cancer, silence of CPT1C induced cell cycle arrest

• Hypoxia adaptation:

  • CPT1C expression is transcriptionally activated by hypoxia-inducible factor-1α (HIF1α)

  • This adaptation helps cancer cells survive under metabolic stress conditions

• Immunosuppression through cancer-associated fibroblasts (CAFs):

  • CPT1C+ CAFs impair tumor immunity by secreting IL-6

  • This induces immunosuppressive M2-like phenotype of macrophages in gastric cancer

  • High CPT1C+ CAFs correlate with enrichment of immunosuppressive cell types

Why might I observe inconsistent CPT1C molecular weights across different tissues or experimental conditions?

Several factors may contribute to the observed variability (70-90 kDa range):

• Species-specific variations:

  • Human CPT1C: Typically observed at 82 kDa

  • Mouse/rat CPT1C: May appear at 70-82 kDa

• Post-translational modifications:

  • Phosphorylation, glycosylation, or other modifications can alter migration patterns

  • CPT1C interacts with SACM1L in a malonyl-CoA dependent manner, which may affect its structure

• Alternative splicing:

  • Different isoforms may be expressed in different tissues

  • Brain-specific processing may result in different molecular weights

• Technical considerations:

  • Gel percentage and running conditions can affect apparent molecular weight

  • Sample preparation methods (heating temperature/time) may affect protein conformation

When troubleshooting, comparison with verified positive controls (human brain tissue for 82 kDa, mouse brain for 70-82 kDa) is recommended .

How can I optimize immunoprecipitation protocols with CPT1C antibodies?

For optimal immunoprecipitation results:

• Sample preparation:

  • Use 0.4-1 mg of protein for each IP reaction

  • RIPA or NP-40 based lysis buffers work well for CPT1C extraction

• Antibody amount optimization:

  • For polyclonal antibodies (e.g., 12969-1-AP): Use 0.5-4.0 μg antibody per 1.0-3.0 mg total protein

  • For other antibodies: 2-4 μg is typically sufficient

  • Incubate overnight with orbital agitation at 4°C

• Pull-down conditions:

  • Use 80-100 μl of Protein-A sepharose beads

  • Incubate for 2-3 hours

  • Wash precipitated complexes with lysis buffer three times

  • Elute with 2× SB/5 mM DTT sample buffer, heated 10 min at 76°C

• Controls:

  • Always include a negative control (non-specific IgG)

  • Consider using brain tissue from CPT1C knockout mice as a negative control

  • Reserve 10% of total protein as input samples before adding antibodies

What are promising research avenues for CPT1C inhibition in cancer therapy?

Given CPT1C's role in cancer progression, several therapeutic strategies warrant investigation:

• Targeting CPT1C in combination with existing therapies:

  • CPT1C overexpression has been linked to rapamycin resistance in tumors

  • Inhibiting CPT1C may enhance efficacy of mTOR inhibitors

• Disrupting CPT1C-mediated fatty acid oxidation:

  • Etomoxir treatment has been shown to restrict FAO rate increases caused by CPT1C

  • Developing more specific CPT1C inhibitors could target cancer metabolism

• Targeting CPT1C+ cancer-associated fibroblasts:

  • CPT1C+ CAFs shape immunosuppressive tumor microenvironments

  • May improve response to immunotherapy in gastric cancer patients

• Exploiting CPT1C's role in AMPAR trafficking:

  • Understanding the dual role of CPT1C in metabolism and receptor trafficking

  • Could provide novel approaches for cancers with neurological manifestations

Future research should focus on developing specific inhibitors of CPT1C that minimize off-target effects, particularly on other CPT1 isoforms that are more broadly expressed.

How can we better understand the distinct roles of CPT1C in neurons versus cancer cells?

This represents an important knowledge gap requiring methodological approaches:

• Tissue-specific conditional knockout models:

  • Generate models with CPT1C deletion in specific tissues/cell types

  • Compare metabolic and signaling changes between neuronal and cancer contexts

• Domain-specific mutations:

  • Create mutations affecting either thioesterase activity or malonyl-CoA binding

  • Test effects on different functions in neurons vs. cancer cells

  • Example mutations (H470A, S252A, D474A) have been used to study catalytic function

• Interactome analysis:

  • Compare CPT1C binding partners in neurons versus cancer cells

  • May reveal context-specific functions and regulatory mechanisms