Recombinant Human gAcrp30

Recombinant Human gAcrp30 is widely used in research because it is the biologically active, globular domain of adiponectin, a key adipokine involved in regulating metabolism, insulin sensitivity, inflammation, and energy homeostasis. Using recombinant gAcrp30 allows for controlled, reproducible studies of its molecular and cellular effects in various experimental systems.

Key reasons to use Recombinant Human gAcrp30 in research applications:

• Metabolic Regulation: gAcrp30 decreases hyperglycemia and reverses insulin resistance, making it essential for studies on diabetes, obesity, and metabolic syndrome. It stimulates AMPK phosphorylation and activation in liver and skeletal muscle, enhancing glucose utilization and fatty acid oxidation.
• Fatty Acid Metabolism: It promotes fat loss by signaling muscle to absorb and burn free fatty acids, and reduces plasma-free fatty acids and glucose levels in animal models.
• Anti-inflammatory and Anti-atherogenic Effects: gAcrp30 antagonizes TNF-α, inhibits NF-κB signaling, and negatively regulates inflammatory responses, supporting research in cardiovascular disease and chronic inflammation.
• Cellular and Molecular Mechanisms: It is involved in cell growth, angiogenesis, tissue remodeling, and can bind/sequester various growth factors, making it valuable for studies in cell signaling, tissue engineering, and cancer biology.
• Receptor Studies: The identification of specific receptors (AdipoR1 and AdipoR2) for gAcrp30 enables mechanistic studies on receptor-mediated signaling pathways in different tissues.
• Disease Models: Recombinant gAcrp30 is used in vitro and in vivo to model and dissect its roles in insulin resistance, obesity, cardiovascular diseases, diabetes mellitus, and inflammation.

Experimental advantages:

• Defined, consistent reagent: Recombinant production ensures batch-to-batch consistency and eliminates variability from serum-derived proteins.
• Species specificity: Human gAcrp30 is ideal for studies using human cells or translational models.
• Functional studies: Enables direct assessment of gAcrp30’s effects on target cells, tissues, or animal models, including dose-response and mechanistic analyses.

Common applications include:

• Metabolic pathway analysis (e.g., AMPK activation, glucose uptake assays)
• Inflammatory response modulation
• Adipocyte and muscle cell signaling studies
• Animal models of metabolic disease
• Screening for therapeutic agents targeting adiponectin pathways

In summary, Recombinant Human gAcrp30 is a critical tool for dissecting the molecular mechanisms of metabolic regulation, insulin sensitivity, and inflammation, and for developing therapeutic strategies for metabolic and cardiovascular diseases.

Yes, you can use recombinant human gAcrp30 as a standard for quantification or calibration in your ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common and accepted practice in quantitative ELISA protocols.

Key considerations and supporting details:

• Purity and Characterization: The recombinant gAcrp30 should be highly purified (≥98% by SDS-PAGE or HPLC is typical) to ensure accuracy in quantification. Purity is essential to avoid interference from contaminants.

• Standard Curve Preparation: For quantitative ELISA, a standard curve must be generated using known concentrations of the target protein standard (here, recombinant gAcrp30). This allows you to interpolate the concentration of gAcrp30 in your unknown samples.

• Formulation: Recombinant gAcrp30 is available in carrier-free or BSA-containing formulations. Carrier-free protein is preferred if BSA or other additives could interfere with your assay, while BSA-containing formulations may enhance stability for routine ELISA use.

• Reconstitution and Handling: If your recombinant protein is lyophilized, follow the manufacturer’s reconstitution instructions carefully, as these may be lot-specific and affect the final concentration.

• Validation: It is important to validate that the recombinant gAcrp30 standard behaves similarly to the native protein in your assay system. This includes confirming linearity, parallelism, and recovery in your matrix of interest.

• Documentation: Always document the source, lot number, and preparation details of your standard for reproducibility and troubleshooting.

Summary Table: Key Requirements for Using Recombinant gAcrp30 as an ELISA Standard

In summary: Recombinant human gAcrp30 is suitable as a standard for ELISA quantification if it is highly purified, accurately quantified, and validated for your specific assay conditions.

Recombinant Human gAcrp30 (globular adiponectin) has been validated in published research for several key applications, primarily in cell-based bioassays, studies of metabolic regulation, endothelial cell biology, and gene expression analysis.

Validated Applications in Published Research:

• Cell-based bioassays:
  Recombinant gAcrp30 has been used to assess its biological activity in vitro, including inhibition of cell proliferation (e.g., mouse M1 myeloid cells), modulation of chemokine induction, and functional rescue of endothelial progenitor cells derived from diabetic patients.

• Metabolic regulation studies:
  It has been applied to investigate its anti-diabetic and anti-atherogenic effects, such as decreasing hyperglycemia, reversing insulin resistance, and promoting fatty acid oxidation in muscle cells.

• Endothelial cell biology:
  gAcrp30 has been used to study its protective effects on human microvascular retinal endothelial cells under hyperglycemic conditions, including suppression of reactive oxygen species (ROS), apoptosis, improvement of cell migration, and barrier function. These studies often employ real-time PCR arrays to profile gene expression related to angiogenesis, extracellular matrix, and adhesion molecules.

• Gene expression analysis:
  Researchers have used gAcrp30 to modulate gene expression in endothelial cells, employing RT-PCR arrays to evaluate changes in genes involved in endothelial function, angiogenesis, and cell adhesion.

• Stem cell niche regulation:
  There is evidence for its use in regulating bone marrow mesenchymal stem cell niches, indicating applications in hematopoietic stem cell research.

Experimental Contexts and Sample Types:

• Whole cell cultures (mouse and human)
• Endothelial cells (including human microvascular retinal endothelial cells)
• Mesenchymal stem cells

Typical Assay Formats:

• Bioassays for cell proliferation and metabolic activity
• ELISA standards for quantification
• RT-PCR and gene expression profiling
• Cell migration and barrier function assays

Summary Table of Validated Applications

Additional Notes:

• The protein is often used at concentrations reflecting physiological levels (e.g., 5–30 μg/mL).
• Carrier-free formulations are preferred when BSA could interfere with downstream applications, such as sensitive cell culture or gene expression studies.
• Most studies are for research use only and not for diagnostic or therapeutic procedures.

If you require protocols or specific experimental details for any of these applications, please specify the context or assay type.

To reconstitute and prepare Recombinant Human gAcrp30 protein for cell culture experiments, follow these general guidelines based on manufacturer recommendations and best practices:

Reconstitution Instructions

1. Centrifuge the vial before opening to ensure all lyophilized powder is at the bottom.
2. Reconstitute the protein with sterile, cold buffer. Most manufacturers recommend:
   • 5 mM Tris, 0.75 mM DTT, pH 8.0 (e.g., Abcam, Novus, Abgenex).
   • Some protocols suggest sterile distilled water or 5 mM Tris-HCl, pH 8.0, with or without DTT.
   • For a final concentration, reconstitute at 0.1–1 mg/mL (e.g., 100 µg protein in 100–1000 µL buffer).
3. Gently pipette the buffer down the sides of the vial to dissolve the protein. Do not vortex to avoid denaturation.
4. Allow the protein to dissolve at room temperature for 15–30 minutes, or on ice if preferred.

Preparation for Cell Culture

1. Aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles.
2. Add carrier protein (e.g., 0.1% BSA or HSA) if the protein is carrier-free and will be stored for more than a few days, as this helps stabilize the protein.
3. Store aliquots at –20°C to –80°C for long-term storage. For short-term use (within a few days), store at 4°C.
4. Thaw aliquots on ice before use and avoid repeated thawing.

Additional Notes

• Filter sterilize the solution if necessary for cell culture (0.22 µm filter).
• Check the specific datasheet for your protein, as some formulations may require additional components (e.g., CaCl₂, NaCl).
• For cell culture experiments, use the protein at the recommended working concentration (typically 0.1–10 µg/mL, depending on the assay).

Example Protocol

• Centrifuge vial (5 min, 1000 × g).
• Add 100 µL of 5 mM Tris, 0.75 mM DTT, pH 8.0 to 100 µg protein.
• Gently mix and let dissolve for 15–30 min at room temperature.
• Aliquot and store at –80°C.
• Thaw on ice before use.

Always refer to the specific product datasheet for any unique requirements or recommendations.