CARTPT Antibody

What is CARTPT and why is it important in neuroscience research?

CARTPT (CART prepropeptide) is a 116-amino acid protein that belongs to the CART family with a molecular weight of approximately 13 kDa. It has gained significant importance in neuroscience research since its discovery in 1981 . CARTPT is involved in multiple physiological processes including appetite regulation, reward mechanisms, and potentially anxiety and depression . The peptide is particularly relevant to studies on feeding behavior, obesity, and addiction due to its expression in brain regions associated with these functions . Recent research has identified GPR160 as a potential receptor for CART, opening new avenues for investigation into CART-mediated signaling pathways .

What are the primary applications for CARTPT antibodies in research?

CARTPT antibodies are primarily utilized in Western Blot (WB), ELISA, immunocytochemistry (ICC), and immunohistochemistry (IHC) applications . They serve as valuable tools for studying CARTPT expression and distribution in various tissues, particularly in the central nervous system. Beyond basic protein detection, these antibodies have been instrumental in functional studies where they can be used to neutralize endogenous CART activity. For example, researchers have injected CART antibodies into the nucleus tractus solitarius (NTS) to block endogenous CART signaling, demonstrating that this intervention increases food intake in fed rats but not in fasted rats . This approach has provided critical insights into CART's physiological role in satiation mechanisms.

What tissues and species show CARTPT expression that can be detected with antibodies?

CARTPT expression has been documented across multiple species including humans, mice, and rats . The protein is expressed in various tissues, with particularly notable expression in:

• Neuronal tissues: various brain regions including the hypothalamus, nucleus accumbens shell, amygdala, hippocampus, and dorsal vagal complex

• Spinal cord: especially in laminae I-III of the dorsal horn

• Peripheral tissues: adrenal medulla, anterior and posterior pituitary

• Cell lines: SH-SY5Y cells have been documented to express CARTPT at detectable levels

Research has shown that CARTPT antibodies can successfully detect the protein in all these tissues, making them versatile tools for comparative studies across different organ systems and species .

What controls should be included when using CARTPT antibodies in functional blocking studies?

When designing experiments using CARTPT antibodies for functional blocking studies, several critical controls should be incorporated:

• Neutralization control: Pre-incubate the CART antibody with CART peptide (typically for 24 hours) to neutralize the antibody before administration. This control has been shown to prevent the stimulatory effect of the antibody on feeding behavior, confirming specificity .

• Knockout validation: Include samples from CART knockout models. For instance, viral-mediated CART knockdown in vagal afferent neurons (VANs) has been demonstrated to abolish the hyperphagic effects of CART antibody injections into the NTS, confirming that the antibody specifically inhibits endogenous CART from vagal sources .

• Concentration gradient: Employ a dose-response approach, as research has shown that CART antibody injection into the NTS dose-dependently increases food intake, with the highest dose (2 ng/mL) resulting in up to 3-fold increase in food intake 2 hours post-injection .

• Physiological state control: Test the antibody effects under different physiological states, as CART antibody-mediated hyperphagia occurs in the fed state when VAN CART expression is high, but not in the fasted state when CART expression is reduced .

What methodological considerations are important when using CARTPT antibodies for immunohistochemistry?

When conducting immunohistochemistry with CARTPT antibodies, researchers should consider several methodological factors:

• Tissue preparation: Proper fixation protocols are essential, with paraformaldehyde fixation being commonly employed. The localization studies that mapped GPR160-like immunoreactivity (the CART receptor) in rat central nervous system used validated commercially available antibodies .

• Validation approaches: It's recommended to validate immunohistochemical findings with complementary techniques such as in situ hybridization or RNAscope. Recent research has employed snRNAseq and RNAscope approaches to confirm antibody specificity and cellular colocalization .

• Cellular resolution: CART and its receptor GPR160 appear to be present on both neurons and glia, requiring careful analysis of cellular distribution. High-resolution imaging techniques may be necessary to distinguish between neuronal and glial expression .

• Region-specific optimization: Given the differential expression of CART across brain regions (with particularly dense expression reported in the amygdala), optimization of staining protocols may need to be region-specific .

• Cross-validation: When possible, use multiple antibodies targeting different epitopes of CART to confirm staining patterns and reduce the risk of false positives.

What are common issues with CARTPT antibody specificity and how can they be addressed?

Addressing specificity concerns with CARTPT antibodies requires systematic validation:

• Multiple detection methods: Researchers should validate CARTPT detection using complementary techniques such as immunohistochemistry, Western blotting, and mRNA analysis. Studies have confirmed that the distribution of CART-encoding mRNA in rat brain matches that identified by polyclonal antibodies raised against peptide fragments predicted from gene sequencing work .

• Knockout controls: Utilizing CART knockout models provides definitive validation. Successful identification of CART KO and WT littermates has been achieved using polymerase chain reaction and immunohistochemistry procedures . Research groups have developed transgenic rat lines harboring floxed Gpr160 genes (Gpr160flx/flx) to examine consequences of site-specific receptor knockout .

• Peptide competition: Pre-incubation of the antibody with purified CART peptide should eliminate specific staining. This approach has been successfully employed to validate antibody specificity in functional studies .

• Addressing post-translational modifications: CART undergoes tissue-specific processing. Research has identified the importance of prohormone convertases PC2 and PC1/3 in CART processing . Antibodies may detect different processed forms, explaining the observation of molecular weights ranging from 4-14 kDa despite a calculated molecular weight of 13 kDa .

• Consideration of genetic polymorphisms: Human studies have identified several polymorphisms in the CART gene that may affect peptide processing and activity. For instance, the Leu34Phe and ALA-156GLY polymorphisms have been associated with obesity, potentially due to deficits in fully processed and bioactive peptide .

How should researchers optimize storage and handling of CARTPT antibodies?

For optimal performance of CARTPT antibodies, the following storage and handling protocols are recommended:

• Storage temperature: Store antibodies at -20°C, where they remain stable for one year after shipment .

• Buffer composition: CARTPT antibodies are typically stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoting considerations: For -20°C storage, aliquoting is generally unnecessary, which simplifies handling procedures .

• BSA content: Some antibody preparations (20μl sizes) contain 0.1% BSA, which should be considered when designing experiments sensitive to BSA presence .

• Freeze-thaw cycles: While specific data for CARTPT antibodies is not provided, it's generally recommended to minimize freeze-thaw cycles to preserve antibody activity.

• Working dilution preparation: When preparing working dilutions, use fresh, sterile buffers and maintain cold chain to preserve antibody functionality.

How can CARTPT antibodies be used to investigate the gut-brain axis in obesity research?

CARTPT antibodies have proven instrumental in elucidating the role of CART signaling in the gut-brain axis and its implications for obesity:

• Functional blocking studies: Injection of CART antibodies into the nucleus tractus solitarius (NTS) increases food intake in fed rats but not in fasted rats, demonstrating CART's role in satiation . This approach can be used to study how different diet interventions affect CART-mediated satiety mechanisms.

• Investigation of vagal afferent neurons: CART expression in vagal afferent neurons (VANs) is regulated by feeding state, with higher expression in fed versus fasted conditions. CART knockdown in these neurons leads to hyperphagia and weight gain by chronically increasing ingestion rate and meal size . Researchers can use CART antibodies to track these changes in expression levels.

• Examination of diet-induced obesity mechanisms: High-fat high-sugar (HFHS) diet reduces CART concentration in nodose ganglia and blunts CCK-induced satiety, correlating with reduced CCKa receptor expression . Antibodies can be employed to quantify these expression changes in different experimental conditions.

• Study of hormonal interactions: CART is required to mediate CCK-induced satiety. Reduced nodose ganglia CART expression coincides with reduced CCK-induced c-Fos in the NTS in HFHS-fed rats . Dual-labeling studies with CART antibodies and other markers can reveal these interaction networks.

• Mechanistic investigations using viral vectors: Combining CART antibodies with viral-mediated CART knockdown approaches can help determine the source and functional relevance of CART signaling in different segments of the gut-brain axis .

What role do CARTPT antibodies play in investigating sex differences in neurobiological research?

Recent research has revealed important sex differences in CART function that can be investigated using CARTPT antibodies:

• Sex-specific behavioral phenotypes: Studies using CART knockout mice have demonstrated sexually dimorphic effects of CART in binge drinking behaviors . Antibodies can be used to map sex-specific expression patterns that might underlie these behavioral differences.

• Hormonal interactions: Given CART's expression in endocrine tissues including the pituitary and adrenal medulla , CART antibodies can help investigate potential interactions between CART signaling and sex hormones that might contribute to sexually dimorphic phenotypes.

• Regional expression differences: Immunohistochemical studies using CART antibodies can reveal potential sex differences in CART expression across different brain regions, particularly those involved in reward processing and addiction-related behaviors .

• Developmental trajectory analysis: CART antibodies can be employed to track the developmental emergence of sex differences in CART expression, potentially revealing critical periods for the organization of sexually dimorphic circuits.

• Mechanistic validation: When combined with genetic approaches such as conditional knockouts, antibodies can help validate the functional relevance of observed sex differences in CART expression to behavioral phenotypes .

How should researchers interpret variations in CARTPT molecular weight observed in Western blots?

The interpretation of varying molecular weights for CARTPT in Western blot analyses requires consideration of several biological factors:

• Post-translational processing: CARTPT undergoes tissue-specific processing. The calculated molecular weight for the full protein is 13 kDa (116 amino acids), but observed weights typically range from 4-14 kDa . This variation reflects different processed forms of the peptide generated by prohormone convertases PC2 and PC1/3 .

• Species differences: While the antibody may show reactivity with human, mouse, and rat samples , slight differences in processing between species may result in different banding patterns.

• Tissue-specific processing: Research has established that tissue-specific processing of the CART preprohormone occurs , which may result in different molecular weight forms being detected in different tissues.

• Experimental conditions: Sample preparation methods, including protein extraction protocols and reducing conditions, can affect the observed molecular weight.

• Polymorphisms: Human studies have identified polymorphisms that may affect peptide processing. For instance, research indicates that certain polymorphisms in the human CART gene can result in deficits in fully processed and bioactive peptide .

When multiple bands are observed, researchers should consider analyzing tissues from CART knockout animals as negative controls and potentially using mass spectrometry to confirm the identity of the observed protein forms.

How can researchers reconcile contradictory findings between CARTPT antibody studies and genetic approaches?

Resolving contradictions between antibody-based and genetic studies of CARTPT requires systematic methodological consideration:

• Antibody specificity validation: Ensure antibodies are validated using multiple approaches including peptide competition, knockout controls, and comparison with mRNA distribution. Studies have confirmed that the distribution of CART-encoding mRNA in rat brain matches that identified by polyclonal antibodies .

• Partial versus complete knockdown: Consider the degree of CART reduction achieved. Research shows that even partial loss of CART in nodose ganglia is sufficient to cause hyperphagia and weight gain , suggesting that varying levels of knockdown might produce different phenotypes.

• Developmental compensation: Germline knockouts may develop compensatory mechanisms absent in acute antibody blocking studies. This might explain why acute CART antibody administration increases food intake while some genetic studies show different phenotypes.

• Species differences: Research in humans has identified polymorphisms associated with obesity , while the situation in experimental animals appears to be more complex. For instance, human studies have associated CART mutations with anxiety and depression, while in rodents, CART administration increases anxiety-like behaviors .

• Site-specific effects: The behavioral effects of CART (orexigenic versus anorexigenic) may depend upon the exact site of administration . Similarly, global versus site-specific conditional knockdown approaches may yield different results.

• Temporal considerations: Acute antibody blocking versus chronic genetic manipulation may reveal different aspects of CART function. The timing of intervention relative to developmental windows might be crucial for certain phenotypes.

To reconcile contradictory findings, researchers should consider employing both approaches in the same study, using site-specific, conditional knockdown methods alongside carefully controlled antibody blocking experiments.

How might CARTPT antibodies contribute to developing therapeutic approaches for obesity?

CARTPT antibodies can facilitate obesity therapeutic development through several research approaches:

• Target validation: CARTPT antibodies help validate the role of CART in energy homeostasis. Research has shown that CART knockdown in vagal afferent neurons increases body weight by up to 20% within four weeks, with concurrent 17% increase in daily food intake . These findings support CART signaling as a potential therapeutic target.

• Mechanism elucidation: Antibodies assist in deciphering the exact signaling pathways through which CART mediates satiety. Studies demonstrate that CART is required for CCK-induced satiation, as CART knockdown abolished satiation induced by exogenous CCK administration . Understanding these interactions may reveal additional therapeutic targets.

• Diet-induced obesity research: CART antibodies help investigate how high-fat high-sugar diets affect satiety mechanisms. Research shows reduced CART concentration in nodose ganglia of HFHS-fed rats coincides with 51% reduction in CCKa receptor mRNA expression . This insight suggests potential interventions to restore normal satiety signaling.

• Polymorphism studies: Human studies using antibodies to study CART expression have identified several polymorphisms associated with obesity. For instance, the −368T>C mutation and the ALA-156GLY polymorphism predispose individuals to obesity . These genetic insights may help identify at-risk populations for targeted interventions.

• Receptor-based therapeutics: With the recent identification of GPR160 as a potential CART receptor , antibodies can help map the distribution of this receptor and evaluate its potential as a drug target. Immunohistochemical mapping of GPR160 has already identified expression in brain regions relevant to feeding behavior .

What emerging technologies might enhance the utility of CARTPT antibodies in neuroscience research?

Several cutting-edge approaches are poised to expand the applications of CARTPT antibodies in neuroscience:

• Conditional knockout models: Newly developed transgenic rat lines harboring floxed Gpr160 genes (Gpr160flx/flx) enable site-specific receptor knockout studies . When combined with antibody-based approaches, these models allow precise dissection of CART signaling in specific neural circuits.

• Single-cell transcriptomics: Integration of CART antibody staining with RNAscope and single-nucleus RNA sequencing (snRNAseq) technologies enables cellular colocalization studies at unprecedented resolution . This approach can reveal which specific cell types express CART and its receptor.

• Viral vector technologies: AAVshRNA approaches targeting GPR160 in selective brain sites, combined with antibody-based validation, allow for examination of CART-driven behaviors with precise spatial control .

• Cell-type specific manipulations: Recent findings suggesting CART receptor GPR160 is present on both glia and neurons open avenues for cell-type specific studies using CART antibodies in combination with cell-type specific markers.

• In vivo imaging: Development of techniques for in vivo imaging of CART expression or activity, potentially using labeled antibodies or antibody-based reporters, could enable real-time tracking of CART dynamics in response to physiological stimuli.

• Multiplex immunohistochemistry: Advanced multiplex staining protocols allow simultaneous detection of CART and multiple interacting proteins, providing comprehensive mapping of CART signaling networks across brain regions.

These technological advances, when combined with traditional antibody-based approaches, promise to significantly expand our understanding of CART's functions in normal physiology and disease states.