Acrp30 Mouse, HEK

What is mouse Acrp30/Adiponectin and what are its primary functions?

Mouse Adiponectin (also known as Acrp30, ADIPOQ, Acdc, Apm1) is an important adipokine involved in regulating fat metabolism and insulin sensitivity. It exhibits direct anti-diabetic, anti-atherogenic, and anti-inflammatory activities within biological systems. The protein functions by stimulating AMPK phosphorylation and activation in liver and skeletal muscle tissues, which enhances glucose utilization and fatty-acid combustion processes. Adiponectin also antagonizes TNF-alpha through negative regulation of its expression in various tissues including liver and macrophages, while counteracting its inflammatory effects. Additionally, it inhibits endothelial NF-kappa-B signaling through a cAMP-dependent pathway . This multifunctional protein may also participate in cell growth, angiogenesis, and tissue remodeling by binding and sequestering various growth factors with distinct binding affinities that depend on the type of complex formed (LMW, MMW, or HMW) .

What is the molecular structure of mouse Acrp30/Adiponectin protein?

The mouse ADIPOQ protein consists of 247 amino acids (Met 1-Asn 247), though commercially available recombinant proteins are often produced as a truncated form encompassing amino acids 18-247 (Glu18-Asn247) . The amino acid sequence includes specific regions that contribute to its multimerization capacity. The mouse Adiponectin protein exists in multiple oligomeric forms, including low molecular weight (LMW), medium molecular weight (MMW), and high molecular weight (HMW) complexes, each with distinct biological activities . The full amino acid sequence contains characteristic patterns of glycine and proline residues that form collagen-like domains, as well as a C-terminal globular domain. The recombinant protein expressed in HEK293 cells has a theoretical molecular weight of approximately 30 kDa by SDS-PAGE, but forms higher-order structures of approximately 180 kDa (hexamer) when analyzed by size exclusion chromatography .

Why choose mouse Acrp30/Adiponectin expressed in HEK293 cells for research?

Mouse Adiponectin expressed in mammalian HEK293 cells provides significant advantages for research applications due to proper post-translational modifications (PTMs) that are essential for biological activity. The HEK293 expression system ensures appropriate protein folding, glycosylation patterns, and formation of higher-order multimeric structures that closely resemble the native protein . Unlike bacterial expression systems, HEK293-expressed Adiponectin maintains critical structural features including the formation of disulfide bonds and appropriate oligomerization into biologically relevant complexes. For research requiring functional studies, the HEK293-expressed protein demonstrates verified bioactivity, including the ability to induce phosphorylation of acetyl-CoA carboxylase (ACC) in C2C12 cells . This expression system typically yields protein with high purity (≥95%) that is suitable for a wide range of experimental applications including Western blotting and functional bioactivity assays .

What expression systems and tags are commonly used for mouse Acrp30/Adiponectin production?

The predominant expression system for producing research-grade mouse Adiponectin is the HEK293 mammalian cell line. This system is preferred due to its ability to generate properly folded protein with appropriate post-translational modifications. Various affinity tags are employed to facilitate purification, with the most common being polyhistidine (His) tags and FLAG tags . The His-tagged version typically includes a C-terminal polyhistidine sequence added to the mouse ADIPOQ protein (Met 1-Asn 247) . Alternatively, FLAG-tagged versions may incorporate the tag at the N-terminus of the truncated protein sequence (aa 16-244) . These affinity tags enable efficient purification through single-step affinity chromatography while generally maintaining the protein's biological activity. The choice between His-tag and FLAG-tag versions depends on the specific experimental requirements, with FLAG tags being particularly useful for immunoprecipitation experiments or when antibody detection is critical. Both tagged versions typically demonstrate high purity (>95% by SDS-PAGE) following appropriate purification protocols .

What are the optimal storage and reconstitution methods for lyophilized recombinant mouse Acrp30/Adiponectin?

For optimal stability and activity preservation, lyophilized recombinant mouse Adiponectin should be stored at -20°C or -80°C until reconstitution. When reconstituting the protein, it is critical to use endotoxin-free water to prevent contamination that could interfere with downstream biological assays. For example, a recommended reconstitution protocol involves adding 50 μl of endotoxin-free water to the lyophilized protein to achieve a concentration of 1 mg/ml . After reconstitution, the protein solution should be handled with care to minimize freeze-thaw cycles, as repeated freezing and thawing can compromise protein structure and activity. For short-term storage (1-2 weeks), reconstituted protein can be kept at 4°C, while aliquoting and storing at -20°C or -80°C is recommended for longer-term storage. The reconstitution buffer composition is typically based on the lyophilization formulation, which often contains 30 mM Tris-Cl, pH 8.0 with 100 mM NaCl to maintain protein stability . Researchers should verify the specific storage recommendations provided by the manufacturer, as optimal conditions may vary slightly between different commercial preparations.

How can researchers verify the quality and activity of purified mouse Acrp30/Adiponectin?

Comprehensive quality assessment of recombinant mouse Adiponectin involves multiple analytical methods. Purity should be evaluated using SDS-PAGE, with high-quality preparations typically showing ≥95% purity . Western blotting with specific anti-Adiponectin antibodies confirms the identity of the protein and can detect both monomeric (~30 kDa) and multimeric forms. Size exclusion chromatography is essential for characterizing the oligomeric state distribution, as functional Adiponectin forms hexamers (~180 kDa) and higher-order structures . For activity verification, a functional bioassay measuring the phosphorylation of acetyl-CoA carboxylase (ACC) in C2C12 cells following treatment with Adiponectin (typically at 25 μg/ml for 15 minutes after serum starvation) provides direct evidence of biological functionality . The phosphorylation can be detected using anti-phospho-ACC antibodies in Western blotting. Additionally, endotoxin testing using the LAL (Limulus Amebocyte Lysate) assay is critical, especially for in vitro and in vivo applications, with high-quality preparations containing <0.01 EU/μg purified protein . Researchers should also assess batch-to-batch consistency by comparing activity metrics across multiple production lots.

How is mouse Acrp30/Adiponectin detected in experimental samples?

Mouse Adiponectin can be detected through multiple complementary methods depending on the experimental context. For protein expression analysis, Western blotting represents a primary approach using specific anti-Adiponectin antibodies, which can detect both monomeric forms (~30 kDa) and multimeric complexes under non-reducing conditions . Immunohistochemistry and immunofluorescence techniques provide spatial information about Adiponectin expression in tissues and cells. For example, in differentiated adipocytes, Adiponectin can be detected using specific antibodies (such as Goat Anti-Mouse Adiponectin/Acrp30 Antigen Affinity-purified Polyclonal Antibody) at concentrations of approximately 10 μg/mL, followed by visualization with fluorescently-labeled secondary antibodies . Importantly, Adiponectin staining typically localizes to cell surfaces and cytoplasm in adipocytes. For quantitative measurements in biological fluids or cell culture supernatants, enzyme-linked immunosorbent assays (ELISAs) provide high sensitivity and specificity. Additionally, mass spectrometry-based proteomics approaches can identify and quantify Adiponectin in complex biological samples, offering advantages for detecting post-translational modifications and distinguishing between different oligomeric forms.

What cell culture systems are appropriate for studying mouse Acrp30/Adiponectin function?

Multiple cellular models are suitable for investigating mouse Adiponectin function, each offering distinct advantages for specific research questions. Primary mouse adipocytes provide a physiologically relevant system that expresses and secretes Adiponectin naturally, though they present challenges regarding consistency and maintenance. The 3T3-L1 preadipocyte cell line, which can be differentiated into mature adipocytes, serves as a widely used model for studying Adiponectin expression, secretion, and regulation . These cells recapitulate many aspects of adipocyte biology and respond to stimuli that modulate Adiponectin production. For examining Adiponectin's metabolic effects, C2C12 mouse myoblasts (which can be differentiated into myotubes) provide an excellent system for studying skeletal muscle responses, including AMPK activation and acetyl-CoA carboxylase (ACC) phosphorylation following Adiponectin treatment . Hepatocyte models (primary or cell lines like AML12) are valuable for investigating Adiponectin's effects on liver metabolism. For mechanistic studies of Adiponectin receptor signaling, HEK293 cells transfected with AdipoR1 or AdipoR2 offer a clean background for dissecting specific signaling pathways. When selecting a cellular model, researchers should consider the expression of Adiponectin receptors (AdipoR1, AdipoR2) and relevant downstream signaling components in their system of choice.

What are the appropriate concentrations and treatment conditions for in vitro studies with recombinant mouse Acrp30/Adiponectin?

The optimal concentrations and treatment conditions for in vitro experiments with recombinant mouse Adiponectin vary based on the specific cellular system and readout being measured. For studies examining AMPK pathway activation and metabolic effects in skeletal muscle cells (such as C2C12), concentrations of 10-30 μg/ml are typically effective, with 25 μg/ml demonstrating clear induction of acetyl-CoA carboxylase (ACC) phosphorylation within 15 minutes of treatment following serum starvation for 24 hours . For adipocytes and hepatocytes, similar concentration ranges (5-30 μg/ml) are generally appropriate. The treatment duration depends on the specific cellular response: acute signaling events (phosphorylation) typically occur within minutes to hours (5 minutes to 4 hours), while transcriptional responses may require 6-24 hours of exposure. Prior to Adiponectin treatment, cells are often serum-starved for 8-24 hours to reduce background signaling and enhance sensitivity to the treatment. The choice between globular Adiponectin and full-length Adiponectin should be guided by the research question, as they may activate different receptor subtypes (AdipoR1 vs. AdipoR2) with varying potency. Control experiments should include vehicle controls and, when applicable, a tagged control protein to account for potential tag-specific effects when using tagged recombinant Adiponectin .

How do different oligomeric forms of mouse Acrp30/Adiponectin influence experimental outcomes?

Adiponectin exists in multiple oligomeric forms that exhibit distinct biological activities and receptor affinities, making the oligomeric distribution a critical consideration in experimental design. The low molecular weight (LMW) trimers, medium molecular weight (MMW) hexamers, and high molecular weight (HMW) multimers each activate different downstream signaling pathways with varying efficiencies . The HMW form is generally considered the most biologically active, particularly for insulin-sensitizing effects, while the globular domain (produced by proteolytic cleavage) preferentially activates AdipoR1 and AMPK pathways in skeletal muscle. When designing experiments, researchers should characterize the oligomeric distribution of their recombinant Adiponectin preparation using non-reducing SDS-PAGE or size exclusion chromatography, as commercial preparations may vary in their oligomeric profiles. The storage conditions and experimental handling can significantly impact oligomerization states, with factors such as pH, calcium concentration, and reducing agents influencing the equilibrium between different forms. Temperature-dependent interconversion between oligomeric states can also occur, potentially confounding experimental results. For studies focused on specific oligomeric forms, size fractionation techniques can be employed to enrich for particular species. Importantly, the interpretation of experimental outcomes should account for the possibility that different oligomeric forms may elicit distinct, sometimes opposing, cellular responses through activation of different receptor subtypes and downstream signaling cascades.

What methodological approaches can distinguish between endogenous and exogenous mouse Acrp30/Adiponectin in experimental systems?

Distinguishing between endogenous and exogenous Adiponectin is critical in many experimental contexts, particularly when studying the effects of recombinant protein administration in cells or animals that express Adiponectin naturally. Several complementary approaches can be employed to make this distinction. Tagged recombinant proteins (His-tag, FLAG-tag) provide the most straightforward solution, as they can be specifically detected using tag-specific antibodies in immunoblotting, immunoprecipitation, or immunofluorescence applications . For His-tagged Adiponectin, anti-His antibodies can selectively detect the exogenous protein, while for FLAG-tagged versions, anti-FLAG antibodies serve the same purpose. Alternatively, species-specific Adiponectin antibodies can be used when the exogenous protein is derived from a different species than the experimental system. Another approach involves using isotope-labeled recombinant proteins (such as those containing 15N or 13C) that can be distinguished from endogenous protein through mass spectrometry analysis. For genetic approaches, introducing silent mutations in the recombinant cDNA sequence allows for specific detection of exogenous mRNA using custom-designed primers for qPCR, though this approach detects expression rather than protein levels. When studying Adiponectin with modified post-translational patterns, specific antibodies recognizing these modifications can further differentiate between endogenous and exogenous forms. Each approach has specific advantages and limitations that should be considered based on the experimental context and available resources.

How does DsbA-L localization in the endoplasmic reticulum affect Adiponectin/Acrp30 production and secretion?

DsbA-L (Disulfide-bond A oxidoreductase-Like protein) plays a crucial role in regulating Adiponectin production, assembly, and secretion through its localization in both mitochondria and the endoplasmic reticulum (ER) . The ER localization of DsbA-L is particularly significant as the ER serves as the primary site for Adiponectin folding, post-translational modification, and oligomeric assembly. As an oxidoreductase-like protein, DsbA-L likely facilitates proper disulfide bond formation in Adiponectin, which is essential for the assembly of higher-order oligomeric structures, particularly the biologically active HMW forms. Disruption of DsbA-L function or expression can impair Adiponectin multimerization and secretion, potentially leading to ER stress and activation of the unfolded protein response (UPR). Under conditions of metabolic stress, such as obesity or inflammation, reduced DsbA-L expression correlates with decreased circulating Adiponectin levels, suggesting that DsbA-L is a critical determinant of Adiponectin bioavailability. Experimental manipulation of DsbA-L expression (through overexpression or knockdown approaches) can serve as a valuable tool for investigating the mechanisms regulating Adiponectin production and secretion . Researchers studying Adiponectin biosynthesis should consider monitoring DsbA-L expression and localization alongside Adiponectin measurements to gain a more comprehensive understanding of the regulatory mechanisms at play.

What factors can affect the stability and activity of recombinant mouse Acrp30/Adiponectin in experimental systems?

Multiple factors can compromise the stability and activity of recombinant mouse Adiponectin in experimental settings. Temperature fluctuations represent a significant concern, with repeated freeze-thaw cycles causing oligomeric disruption and potential denaturation. Researchers should aliquot reconstituted protein and avoid more than 1-2 freeze-thaw cycles. The buffer composition significantly impacts stability, with optimal conditions typically including Tris-Cl (pH 8.0) and physiological salt concentrations (approximately 100-150 mM NaCl) . Deviations from the recommended pH range (7.5-8.5) can alter oligomeric distribution and reduce activity. Mechanical stress through excessive vortexing or vigorous pipetting can disrupt higher-order oligomeric structures, so gentle handling techniques are advised. Protease contamination in experimental systems can lead to degradation; therefore, protease inhibitors should be included when working with biological samples. The presence of reducing agents (such as DTT or β-mercaptoethanol) at high concentrations can disrupt critical disulfide bonds, affecting oligomerization and activity. Metal ions, particularly transition metals, can promote oxidation and aggregation, so EDTA (0.1-1 mM) may be included in storage buffers. When working with serum-containing media, serum proteases and binding proteins may interact with recombinant Adiponectin, potentially altering its effective concentration or activity. Additionally, adsorption to plastic surfaces during storage or experimental manipulation can reduce the effective concentration; this can be minimized by including carrier proteins (such as BSA at 0.1%) in very dilute solutions.

How can researchers overcome challenges in detecting mouse Acrp30/Adiponectin multimeric forms?

Detecting and characterizing Adiponectin's multimeric forms presents significant technical challenges that require specialized approaches. Native PAGE or non-reducing SDS-PAGE is essential for preserving oligomeric structures, as standard reducing conditions with β-mercaptoethanol or DTT disrupt the disulfide bonds critical for multimerization. When using Western blotting, gentle transfer conditions (lower voltage, longer time) improve the detection of high molecular weight complexes that transfer less efficiently than monomers. Specialized gradient gels (typically 4-15% or 4-20%) provide better resolution across the wide molecular weight range needed to visualize all oligomeric forms simultaneously. For sample preparation, researchers should avoid boiling samples intended for oligomeric analysis, instead using room temperature or mild heating (37°C) in non-reducing sample buffer. Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) offers superior resolution for characterizing oligomeric distribution in solution. When quantifying different oligomeric forms, densitometry of Western blots should be conducted with caution, as antibody accessibility may vary between different oligomeric structures. For immunoprecipitation of specific oligomeric forms, native conditions throughout the procedure are critical. Crosslinking approaches using agents such as disuccinimidyl suberate (DSS) or glutaraldehyde can stabilize oligomeric forms prior to analysis, though optimization is required to prevent artifactual crosslinking. Additionally, researchers should be aware that the distribution of oligomeric forms can be dynamically influenced by experimental conditions including temperature, pH, and ionic strength.

What technical considerations are important when comparing results across different commercial sources of mouse Acrp30/Adiponectin?

When comparing experimental results obtained using different commercial preparations of mouse Adiponectin, researchers must account for several potential sources of variation. The expression system significantly impacts post-translational modifications and biological activity, with HEK293-derived protein generally offering more consistent activity than E. coli-expressed versions due to proper glycosylation and folding . Different affinity tags (His, FLAG, GST, etc.) can influence protein behavior; His-tagged and FLAG-tagged versions may exhibit subtle differences in receptor binding or signaling activation . The specific protein sequence used can vary between vendors, with some offering full-length protein (Met1-Asn247) while others provide truncated versions (Glu18-Asn247 or similar) , potentially affecting certain functions. The oligomeric distribution profile can vary significantly between sources and lots, with some preparations enriched in HMW forms while others contain predominantly LMW species. Formulation differences, including buffer composition, pH, and additives, may influence stability and activity during storage and experimentation. Endotoxin contamination levels should be verified (ideally <0.01 EU/μg) as even low levels can confound inflammatory response studies . Batch-to-batch variability within the same vendor necessitates lot testing for critical experiments. Validation methods vary between suppliers, with some providing functional bioassay data while others rely solely on purity assessments. To ensure experimental reproducibility, researchers should maintain detailed records of the specific product, lot number, and handling procedures used, and ideally include positive controls when comparing new lots or sources against previously validated material.

How do post-translational modifications affect mouse Acrp30/Adiponectin function in research applications?

Post-translational modifications (PTMs) of mouse Adiponectin critically influence its structure, oligomerization, receptor binding, and signaling properties. Hydroxylation and glycosylation of conserved lysine residues within the collagenous domain are essential for proper folding and assembly of higher-order oligomeric structures, particularly the biologically active HMW forms. These modifications vary depending on the expression system, with mammalian cell systems like HEK293 providing more physiologically relevant PTM patterns than prokaryotic systems . Disulfide bond formation, particularly involving a conserved cysteine residue near the N-terminus, is essential for trimerization and subsequent assembly into hexamers and HMW complexes. The extent of disulfide-mediated oligomerization can be influenced by the redox environment during protein production and storage. Researchers should be aware that different cell types may process PTMs differently, potentially yielding Adiponectin with altered biological activities. For instance, adipose tissue under inflammatory stress may produce Adiponectin with reduced hydroxylation and glycosylation, affecting its oligomerization and function. When selecting recombinant Adiponectin for research, the expression system should be chosen based on the importance of PTMs to the specific research question. For studies focused on receptor binding and signal transduction, HEK293-expressed protein with appropriate PTMs is generally preferred . For structural studies requiring homogeneous preparations, E. coli-expressed domains may be suitable. Importantly, analytical techniques such as mass spectrometry can be employed to characterize the PTM profile of different Adiponectin preparations, providing valuable information for interpreting experimental results.

What are the key parameters for optimizing Western blot detection of mouse Acrp30/Adiponectin?

Optimizing Western blot detection of mouse Adiponectin requires attention to several key parameters that address its unique structural and biochemical properties. Sample preparation represents a critical first step, with different protocols required depending on whether monomeric or oligomeric forms are being targeted. For monomeric detection, standard reducing conditions with heat denaturation (95°C for 5 minutes) in the presence of β-mercaptoethanol or DTT are appropriate. For oligomeric analysis, non-reducing conditions without boiling (room temperature or 37°C incubation) are essential to preserve higher-order structures. Polyacrylamide gel concentration should be selected based on the target forms: 10-12% gels work well for monomers (~30 kDa), while gradient gels (4-15% or 4-20%) provide better resolution for the full range of oligomeric species. The transfer step requires optimization, with semi-dry transfer systems using PVDF membranes generally providing good results for monomeric Adiponectin, while wet transfer at lower voltage for extended periods (30V overnight at 4°C) may improve transfer efficiency of HMW complexes. For antibody selection, primary antibodies raised against mouse Adiponectin, such as Goat Anti-Mouse Adiponectin/Acrp30 Antigen Affinity-purified Polyclonal Antibody, have demonstrated good specificity at concentrations of approximately 10 μg/mL . When detecting tagged versions, antibodies specific to the tag (anti-His, anti-FLAG) can provide cleaner results, especially in complex biological samples. Blocking with 5% non-fat dry milk in TBST typically provides optimal results, though BSA may be preferred when using phospho-specific antibodies to detect downstream signaling events. Extended primary antibody incubation (overnight at 4°C) often improves sensitivity, particularly for complex samples or when antibody affinity is moderate.

Comparative Analysis: Mouse vs. Human Acrp30/Adiponectin in Experimental Systems

Understanding the similarities and differences between mouse and human Adiponectin is crucial for translational research and proper experimental design. While mouse and human Adiponectin share approximately 85% amino acid sequence homology, important structural and functional differences exist that can impact experimental outcomes. The table below summarizes key comparative parameters:

This comparative analysis highlights the importance of selecting the appropriate species-matched Adiponectin for experimental systems to ensure physiologically relevant results, particularly for studies focused on receptor binding, signaling activation, and metabolic responses .

What concentration ranges of mouse Acrp30/Adiponectin are physiologically relevant for in vitro and in vivo studies?

Selecting physiologically relevant concentrations of mouse Adiponectin for experimental studies is essential for generating translatable results. The table below summarizes recommended concentration ranges based on physiological levels and documented effective doses in various experimental systems:

When designing experiments, researchers should consider that the effective concentration may vary based on the specific readout being measured, the cell type or tissue being studied, and the oligomeric composition of the preparation. Dose-response studies are recommended when working with new experimental systems to establish optimal concentration ranges. Additionally, researchers should note that local tissue concentrations may differ significantly from circulating levels, particularly in adipose tissue where local concentrations are substantially higher than serum levels .