CES3 Antibody

What is CES3 and what functional roles does it play in biological systems?

CES3 (Carboxylesterase 3) belongs to a large family of carboxylesterases responsible for hydrolyzing ester and amide bonds. Also known as triacylglycerol hydrolase (TGH), CES3 plays multiple critical roles in metabolism. It functions as a major lipase in white adipose tissue and actively participates in drug metabolism and activation processes . CES3 interacts with lipid biosynthesis pathways, linking lipid storage with breakdown and playing a significant role in maintaining lipid equilibrium throughout the body . Its broad substrate specificity allows it to hydrolyze various compounds ranging from small molecule esters to long-chain fatty acid esters and thioesters . Additionally, CES3 contributes to the detoxification of xenobiotics and the activation of ester and amide prodrugs .

What are the key specifications of commercially available CES3 antibodies?

CES3 antibodies are available for multiple species, with mouse/rat and human variants being most common. For mouse/rat research, antibodies targeting the Tyr19-Glu561 epitope region (Accession # Q8VCT4) are available as affinity-purified polyclonal formulations . For human CES3 research, recombinant monoclonal antibodies targeting regions within the N-terminus to C-terminus are available . Western blot applications typically detect CES3 at approximately 65 kDa for mouse/rat samples and 62 kDa for human samples . These antibodies demonstrate reactivity in multiple applications including Western blotting and immunofluorescence staining, with validated performance in tissues known to express CES3, such as lung tissue and adipose tissue .

What is the optimal storage protocol for preserving CES3 antibody activity?

To maintain optimal CES3 antibody performance, proper storage practices are essential. Manufacturers recommend using a manual defrost freezer and avoiding repeated freeze-thaw cycles that can degrade antibody quality . For long-term storage of unopened antibody, maintain at -20 to -70°C for up to 12 months from date of receipt . After reconstitution, short-term storage (up to 1 month) can be achieved at 2 to 8°C under sterile conditions . For extended storage after reconstitution, temperatures of -20 to -70°C under sterile conditions will maintain activity for up to 6 months . Following these guidelines ensures consistent antibody performance across experiments and maximizes shelf-life.

What are the validated applications for CES3 antibodies in research?

CES3 antibodies have been validated for multiple experimental applications across different tissue and cell types. Western blotting applications have been thoroughly validated for both mouse/rat and human CES3 detection, with confirmed specificity in lung tissue, adipose tissue, and cell lines such as HeLa . Immunofluorescence staining has demonstrated utility for subcellular localization studies, particularly for tracking CES3 recruitment to lipid droplets in response to physiological stimuli such as cold exposure . Co-immunoprecipitation techniques have also successfully employed CES3 antibodies to investigate protein-protein interactions, though notably, no interaction was detected between CES3 and LC3 (an autophagy marker) . These diverse applications make CES3 antibodies versatile tools for investigating carboxylesterase biology across multiple experimental contexts.

What protocol optimizations are recommended for Western blot detection of CES3?

For optimal Western blot detection of CES3, several technical considerations should be addressed. The protocol should be performed under reducing conditions to ensure proper protein denaturation and epitope exposure . For mouse/rat CES3 detection, PVDF membrane is recommended with 1 μg/mL of Mouse Carboxylesterase 3/CES3 Antigen Affinity-purified Polyclonal Antibody, followed by HRP-conjugated Anti-Goat IgG Secondary Antibody . For human samples, a 1/500 dilution of Anti-CES3/TGH antibody followed by Goat Anti-Rabbit IgG H&L (Dylight800) at 1/10000 dilution yields optimal results . Expected band size varies slightly between species: approximately 65 kDa for mouse/rat CES3 and 62 kDa (predicted) for human CES3 . Buffer conditions can significantly impact results; for mouse/rat CES3 detection, Immunoblot Buffer Group 8 has been validated for consistent performance .

How can I assess CES3 subcellular localization using immunofluorescence techniques?

Immunofluorescence (IF) staining offers powerful insights into CES3 subcellular localization and its dynamic changes in response to physiological stimuli. For optimal results, co-staining with organelle-specific markers is recommended. To investigate lipid droplet association, co-staining with perilipin-1 antibodies allows visualization of CES3 recruitment to lipid droplets, where colocalization appears as yellow signals in merged images (red: CES3; green: perilipin-1) . For investigating potential autophagosome association, co-staining with LC3 antibodies can be performed, though research indicates no significant colocalization under β-adrenergic stimulation . Similarly, mitochondrial targeting can be assessed through co-staining with COX IV antibodies, though current evidence suggests CES3 does not localize to mitochondria during cold exposure . These multi-label approaches provide comprehensive spatial information about CES3 dynamics within cellular compartments under different experimental conditions.

What sample preparation techniques maximize CES3 detection sensitivity?

For optimal CES3 detection, sample preparation must preserve protein integrity while ensuring adequate exposure of target epitopes. For tissue samples, preparation of lysates from freshly collected specimens yields superior results compared to frozen samples, particularly for adipose tissue where lipid content can interfere with protein extraction . When fractionating samples to isolate lipid droplets for CES3 localization studies, careful density gradient centrifugation is required to maintain the native association of CES3 with lipid droplets . For immunoprecipitation studies, mild detergent conditions preserve protein-protein interactions while sufficiently solubilizing membrane-associated CES3 . Silver staining techniques coupled with mass spectrometry provide powerful validation of antibody specificity, with successful detection of CES3-specific peptide sequences such as LGIWGFFSTGDEHSR confirming antibody target accuracy .

How does cold exposure affect CES3 distribution and function in adipose tissues?

Cold exposure induces significant changes in CES3 localization within adipose tissues, with important implications for understanding thermogenic responses. Western blotting analysis of lipid droplet fractions from brown adipose tissue (BAT) demonstrates increased CES3 recruitment to lipid droplets during cold exposure compared to room temperature conditions . This recruitment pattern is observed regardless of dietary conditions, occurring in both standard diet and high-fat diet-fed mice . Heat map data further confirms enhanced CES3 recruitment to lipid droplets in subcutaneous white adipose tissue (sWAT) in response to cold . Immunofluorescence co-staining with perilipin-1 visually confirms this redistribution, with increased yellow signals in merged images indicating greater colocalization of CES3 with lipid droplets following cold exposure . This cold-induced recruitment suggests CES3 plays a role in mobilizing lipid stores during thermogenic responses, potentially through enhanced lipolytic activity at the lipid droplet surface.

What interactions exist between CES3 and autophagy or mitochondrial pathways?

Despite CES3's dynamic recruitment to lipid droplets during conditions that stimulate lipolysis, research indicates limited direct interaction with either autophagy machinery or mitochondrial compartments. Coimmunoprecipitation experiments demonstrate that even when CES3 molecules are successfully immunoprecipitated with α-GFP antibody, no LC3 (a key autophagy marker) is detected by α-LC3 antibody, suggesting absence of physical interaction between CES3 and autophagy components under various stimuli . Co-immunofluorescence staining with α-Ces3 and α-LC3 in BAC cells further confirms this finding, with no yellow signal observed in merged images under isoproterenol treatment, indicating CES3 does not localize to autophagosomes during β-adrenergic stimulation . Similarly, co-staining with mitochondrial marker COX IV in cold-exposed subcutaneous white adipose tissue shows no significant yellow signal in merged images, demonstrating that CES3 does not target mitochondria during cold-induced thermogenesis .

What role does CES3 play in drug metabolism and pharmacological responses?

CES3 contributes significantly to drug processing through its hydrolytic activity on ester and amide bonds present in various medications. It shows low catalytic efficiency for hydrolysis of CPT-11 (7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin), a prodrug for camptothecin used in cancer therapeutics . This selective activity profile is important when designing ester-containing drugs, as CES3 activity can affect bioavailability of therapeutic agents . Many medications incorporate ester linkages specifically to improve bioavailability, making CES3's hydrolytic function critical in determining drug efficacy and metabolism . Understanding tissue-specific expression patterns of CES3 can help predict differential drug responses across organs and potentially explain inter-individual variability in drug metabolism . These pharmacological considerations make CES3 an important target for research in drug development and personalized medicine approaches.

How can I validate the specificity of CES3 antibody staining patterns?

Validating CES3 antibody specificity requires multiple complementary approaches to ensure reliable results. Western blot analysis should detect a single band at the expected molecular weight (approximately 65 kDa for mouse/rat CES3, 62 kDa for human CES3) in tissues known to express CES3 . Mass spectrometry validation provides definitive confirmation, with studies demonstrating 85% sequence coverage of CES3 and identification of unique peptide sequences such as LGIWGFFSTGDEHSR that are specific to CES3 . For immunofluorescence applications, comparison of staining patterns across multiple antibodies targeting different CES3 epitopes can confirm pattern consistency . Positive controls should include tissues with established CES3 expression (lung, adipose tissue), while negative controls with secondary antibody only or isotype controls help identify non-specific binding . This multi-faceted approach ensures confidence in the specificity of observed CES3 staining patterns.

What are common pitfalls in CES3 antibody experiments and how can they be avoided?

Several technical challenges can complicate CES3 antibody experiments if not properly addressed. Cross-reactivity with other carboxylesterase family members can occur due to sequence homology (particularly between species variants, with mouse CES3 sharing 93% homology with rat CES3) . This can be mitigated by carefully selecting antibodies raised against unique epitope regions and validating specificity through mass spectrometry . For adipose tissue analysis, high lipid content can interfere with protein extraction and detection, necessitating optimized lysis buffers with appropriate detergent concentrations . Background issues in immunofluorescence can result from non-specific binding, requiring careful blocking optimization and inclusion of appropriate controls . When studying CES3 localization changes in response to stimuli like cold exposure, timing of sample collection is critical, as transient changes may be missed with improper experimental design . Addressing these potential pitfalls through methodological optimization ensures reliable and reproducible CES3 antibody experimental outcomes.

How can antibody engineering enhance CES3 research applications?

Advanced antibody engineering technologies offer promising opportunities for enhancing CES3 research capabilities. Recent developments in antibody selection and design techniques, including high-throughput sequencing and computational analysis, enable the creation of antibodies with customized specificity profiles . These approaches can generate antibodies with either highly specific binding to particular CES3 epitopes or cross-specificity for multiple regions . For CES3 research, engineered antibodies could potentially distinguish between closely related carboxylesterase family members with unprecedented specificity, overcoming current limitations in discriminating highly homologous proteins . Computational models can identify different binding modes associated with particular ligands, allowing researchers to design antibodies with predefined binding characteristics . These engineered antibodies could enable more precise tracking of CES3 in complex tissue environments and potentially distinguish between different functional states of the enzyme.

What is known about CES3's role in metabolic disorders and potential therapeutic applications?

CES3's significant involvement in lipid metabolism positions it as a molecule of interest in metabolic disorder research. Its function as a major lipase in white adipose tissue and its role in maintaining lipid equilibrium suggest potential implications in conditions like obesity, diabetes, and fatty liver disease . The observed recruitment of CES3 to lipid droplets during cold exposure indicates involvement in thermogenic responses and energy expenditure pathways, which are increasingly targeted for metabolic disorder interventions . While direct therapeutic applications targeting CES3 remain to be fully explored, its participation in drug metabolism pathways suggests potential for modulating drug responses through CES3 activity regulation . Further research is needed to clarify how CES3 expression and activity changes contribute to disease progression or protection, and whether CES3 modulation could offer therapeutic benefits in metabolic disorders characterized by dysregulated lipid metabolism.

How does CES3 compare functionally with other carboxylesterase family members?

Understanding CES3's unique functional properties within the broader carboxylesterase family provides important context for targeted research applications. While CES3 shares the fundamental hydrolytic activity characteristic of carboxylesterases, its substrate preferences and tissue distribution patterns distinguish it from other family members . CES3's pronounced role as a major lipase in white adipose tissue suggests specialized function in lipid metabolism compared to other carboxylesterases with broader tissue distribution . Its demonstrated recruitment to lipid droplets during cold exposure reveals dynamic subcellular trafficking not extensively documented for other family members . Regarding drug metabolism, CES3 shows differential catalytic efficiency for various medications, including relatively low activity toward the cancer therapeutic prodrug CPT-11, indicating substrate specificity distinctions from other carboxylesterases . These functional specializations make CES3 particularly relevant for research focused on adipose tissue biology and specific pharmacological applications, while other family members may have greater relevance in different tissue contexts or metabolic pathways.

Table 1: Optimal Storage Conditions for CES3 Antibodies

Table 2: Western Blot Detection Parameters for CES3

Table 3: CES3 Subcellular Localization Under Various Physiological Conditions