CPT1A Antibody, HRP conjugated

What is CPT1A and why is it an important research target?

CPT1A (carnitine palmitoyltransferase 1A) is a liver-type enzyme belonging to the carnitine/choline acetyltransferase family. It plays a critical role in mitochondrial fatty acid oxidation (FAO) by facilitating the transport of long-chain fatty acids into mitochondria. CPT1A has emerged as a significant research target due to its involvement in multiple pathological conditions. It functions as a determining factor for abnormal activation of FAO in nasopharyngeal carcinoma (NPC) cells and has been implicated in disorders like multiple sclerosis. CPT1A deficiency clinically presents as recurrent attacks of fasting hypoketotic hypoglycemia, making it relevant for metabolic research. The gene is located on chromosome 11q13.3, a region frequently amplified in head and neck squamous cell carcinoma (HNSCC), highlighting its potential role in cancer biology .

What are the key differences between HRP-conjugated CPT1A antibody and other conjugates?

The HRP-conjugated CPT1A antibody (CSB-PA005922LB01HU) differs from other conjugates primarily in its application suitability. While the HRP conjugate is optimized for ELISA applications, other available conjugates include FITC (CSB-PA005922LC01HU) for fluorescence-based detection and Biotin (CSB-PA005922LD01HU) for signal amplification in ELISA. Each conjugate maintains specificity for CPT1A but provides different detection modalities. The HRP conjugate offers enzymatic signal amplification through peroxidase activity, making it ideal for colorimetric detection in ELISA with high sensitivity. In contrast, FITC conjugates are preferable for fluorescence microscopy and flow cytometry, while Biotin conjugates allow for secondary detection systems with increased sensitivity through biotin-streptavidin interactions .

How should I determine the optimal working dilution for CPT1A-HRP antibody in my ELISA experiments?

Determining the optimal working dilution for CPT1A-HRP antibody requires systematic titration to maximize signal-to-noise ratio while minimizing background. Begin with a broad range dilution series (e.g., 1:1000, 1:2000, 1:5000, 1:10000) against known positive and negative controls. Plot signal-to-noise ratios against antibody dilution to identify the inflection point that provides maximum specific signal with minimal background. For CPT1A-HRP conjugated antibody in ELISA applications, start with manufacturer-recommended dilutions and adjust based on your specific experimental conditions including antigen concentration, blocking reagents, and detection systems. The optimal dilution should provide reproducible signals that fall within the linear range of detection. It's advisable to perform this optimization for each new lot of antibody and for different experimental matrices (cell lysates, tissue homogenates, serum samples) .

What methodological approaches can determine if CPT1A expression correlates with therapeutic resistance in cancer models?

To determine if CPT1A expression correlates with therapeutic resistance in cancer models, implement a systematic, multi-step experimental approach. First, establish baseline CPT1A expression profiles in sensitive and resistant cancer cell lines using the CPT1A-HRP antibody in ELISA, validated by Western blot with the unconjugated antibody at 1:5000 dilution. Next, develop isogenic resistant models by exposing sensitive cell lines (such as A549, HepG2, or HeLa cells) to increasing concentrations of therapeutic agents while monitoring changes in CPT1A expression. Conduct knockdown and overexpression studies using shCPT1A constructs (as validated in A549 and HepG2 cells) to evaluate whether modulating CPT1A levels alters drug sensitivity. Based on findings that CPT1A regulates cell cycle progression through effects on cyclin D1 and CDK4 expression, assess whether CPT1A-mediated metabolic adaptations contribute to therapy evasion by measuring fatty acid oxidation rates in resistant versus sensitive cells. Finally, analyze clinical samples using immunohistochemistry with CPT1A antibody (1:500-1:2000 dilution) to correlate expression levels with treatment outcomes and recurrence rates. This comprehensive approach can reveal whether CPT1A-dependent metabolic rewiring represents a targetable mechanism of therapeutic resistance .

What are common causes of high background when using CPT1A-HRP antibody in ELISA, and how can these be mitigated?

High background when using CPT1A-HRP antibody in ELISA can stem from multiple sources that require systematic troubleshooting. Insufficient blocking is a primary cause; optimize this by testing different blocking agents (BSA, milk, commercial blockers) at various concentrations (1-5%) and incubation times (1-2 hours at room temperature or overnight at 4°C). Excessive antibody concentration often contributes to non-specific binding; perform a titration series with the CPT1A-HRP antibody starting from 1:1000 up to 1:10000 to identify the optimal signal-to-noise ratio. Inadequate washing represents another common issue; implement stringent washing protocols with at least 5-6 washes per step using PBS-T (0.05-0.1% Tween-20). Cross-reactivity with similar epitopes can be addressed by pre-absorbing the antibody with relevant proteins or using more stringent blocking conditions. Buffer composition matters significantly; optimize pH (typically 7.2-7.4) and ionic strength, and consider adding low concentrations (0.1-0.5%) of irrelevant proteins from the same species as your samples. Finally, substrate incubation conditions affect background development; optimize incubation time and temperature, and protect HRP substrates from light to prevent premature oxidation. Each optimization step should be performed systematically while maintaining appropriate positive and negative controls .

How should I modify protocols when using CPT1A-HRP antibody to detect low abundance CPT1A in clinical samples?

For detecting low abundance CPT1A in clinical samples using CPT1A-HRP antibody, implement several protocol modifications to enhance sensitivity while maintaining specificity. First, optimize sample preparation by using specialized extraction buffers containing phosphatase and protease inhibitors that preserve CPT1A integrity, particularly important given its observed molecular weight of 86 kDa (calculated: 88 kDa). Employ a signal amplification strategy by utilizing a biotin-tyramide system that can increase detection sensitivity by 10-100 fold when combined with HRP conjugates. Consider using extended primary antibody incubation (overnight at 4°C) to maximize antigen capture, and implement a more sensitive substrate system such as chemiluminescent or fluorescent HRP substrates instead of standard colorimetric options. For particularly challenging samples, a sandwich ELISA approach may be beneficial, using a capture antibody targeting a different CPT1A epitope (such as the unconjugated antibody CSB-PA005922LA01HU at 1:500 dilution) followed by detection with the HRP-conjugated version. Additionally, concentrating clinical samples through immunoprecipitation prior to ELISA can significantly enhance detection of low abundance targets. This approach has been successfully employed in studies examining CPT1A expression in liver tissues where protein levels were significantly downregulated in certain experimental conditions .

How does the performance of CPT1A-HRP antibody compare with fluorescent conjugates in multiplexed imaging experiments?

The performance characteristics of CPT1A-HRP antibody versus fluorescent conjugates in multiplexed imaging experiments differ substantially across several parameters. HRP-conjugated antibodies provide enzymatic signal amplification but offer limited multiplexing capability due to the diffuse nature of precipitated substrate products and potential cross-reactivity when using multiple HRP-conjugated antibodies simultaneously. In contrast, fluorescent conjugates such as the CPT1A-FITC (CSB-PA005922LC01HU) or specialized fluorescent antibodies like CoraLite Plus 647-conjugated CPT1A (CL647-66039) with excitation/emission maxima of 654/674 nm enable precise subcellular localization and multi-parameter analysis. The fluorescent conjugates demonstrate superior resolution for co-localization studies but may suffer from photobleaching during extended imaging sessions. For experiments requiring both high sensitivity and multiplexing capability, a hybrid approach may be optimal: use the CPT1A-HRP antibody with tyramide signal amplification (TSA) to detect low-abundance CPT1A followed by fluorescent conjugates for additional markers. When designing multiplexed experiments, consider the spectral overlap between fluorophores, tissue autofluorescence profiles, and available imaging filters. Recent flow cytometry data with CoraLite Plus 647-conjugated CPT1A antibody demonstrates excellent signal separation when combined with other markers, making it preferable for multi-parameter analyses despite the higher sensitivity of HRP-based detection methods .

What criteria should guide the selection between polyclonal and monoclonal CPT1A antibodies for specific research applications?

Selection between polyclonal and monoclonal CPT1A antibodies should be guided by specific experimental requirements and technical considerations. Polyclonal antibodies, such as the rabbit polyclonal CPT1A antibody (CSB-PA005922LA01HU), offer broader epitope recognition, enhancing detection sensitivity particularly for proteins with post-translational modifications or in partially denatured states. This characteristic makes them advantageous for applications like Western blotting (1:2000-1:5000 dilution) and immunohistochemistry (1:20-1:200 dilution). Conversely, monoclonal antibodies, like the mouse monoclonal CPT1A antibody (CL647-66039), provide superior specificity to single epitopes, ensuring consistent lot-to-lot reproducibility critical for longitudinal studies and standardized assays. For quantitative analyses requiring precise epitope targeting, such as monitoring specific phosphorylation states or protein isoforms, monoclonal antibodies are preferred. Consider species compatibility when designing co-staining experiments; the rabbit polyclonal can be paired with mouse-derived antibodies without cross-reactivity concerns. Application-specific factors also matter—for flow cytometry, the monoclonal CPT1A antibody conjugated to fluorescent dyes offers direct single-step staining, while ELISA applications benefit from the high-affinity binding of polyclonal antibodies. When studying CPT1A in complex disease models such as multiple sclerosis or cancer systems where subtle protein variant detection is crucial, the polyclonal antibody's ability to recognize multiple epitopes provides comprehensive target capture .

How can I distinguish between specific CPT1A signals and potential cross-reactivity with other CPT isoforms?

Distinguishing between specific CPT1A signals and potential cross-reactivity with other CPT isoforms (particularly CPT1B and CPT1C) requires implementing multiple validation strategies. First, conduct comparative Western blot analysis using the unconjugated form of the CPT1A antibody (at 1:5000 dilution) against tissue panels with known differential expression of CPT isoforms—liver tissue predominantly expresses CPT1A (86 kDa), skeletal muscle expresses CPT1B (88 kDa), and brain tissue expresses CPT1C (88 kDa). Second, perform parallel assays with genetic validation using shRNA knockdown controls specific to CPT1A, as has been validated in A549 and HepG2 cell lines. When interpreting ELISA results with the HRP-conjugated antibody, analyze signal ratios across these tissue types to identify potential cross-reactivity. Third, conduct peptide competition assays using synthetic peptides corresponding to unique regions of each CPT1 isoform—pre-incubation with CPT1A-specific peptides should abolish specific signals while leaving cross-reactive signals intact. Finally, consider the subcellular localization pattern in immunocytochemistry or immunohistochemistry experiments—CPT1A typically shows mitochondrial outer membrane localization in hepatocytes. For definitive isoform discrimination in crucial experiments, consider supplementing antibody-based detection with isoform-specific PCR to correlate protein and mRNA expression patterns .

How does CPT1A function as a potential therapeutic target in autoimmune neurological diseases?

CPT1A functions as a promising therapeutic target in autoimmune neurological diseases through its central role in fatty acid metabolism and subsequent immunomodulatory effects. Research utilizing CPT1A antibodies has revealed that specific mutations in the CPT1A gene (P479L in Inuits and G710E in Hutterites) correlate with remarkably low prevalence of multiple sclerosis (MS). Mechanistic studies employing a Cpt1a P479L mouse strain demonstrated significantly reduced disease severity in experimental autoimmune encephalomyelitis (EAE), a model of MS, compared to wild-type mice. This protective effect was particularly pronounced when animals were maintained on a high-fat diet (HFD), which exacerbated disease in wild-type mice but not in Cpt1a P479L mice. Wild-type mice on HFD showed significantly higher clinical scores from day 15 onward, reaching highly significant differences (p < 0.0001) in the last six days of the experiment. The therapeutic potential of CPT1A inhibition lies in its ability to modulate T cell metabolism, as CPT1A-mediated fatty acid oxidation influences T cell differentiation and function. By shifting metabolic dependencies away from fatty acid oxidation, CPT1A inhibition may selectively impair pathogenic T cell populations while preserving regulatory T cell function. This metabolic immunomodulation represents a novel approach to autoimmune disease treatment that targets fundamental cellular energetics rather than broad immunosuppression, potentially offering improved specificity and reduced side effects compared to current therapies .

What is the significance of CPT1A amplification in cancer progression and how might this inform personalized medicine approaches?