Recombinant Human Vaspin

Using Recombinant Human Vaspin in research is valuable for studying metabolic regulation, insulin sensitivity, inflammation, and cardiovascular function, particularly in the context of obesity, type 2 diabetes, and related metabolic disorders.

Key scientific reasons to use recombinant human vaspin include:

• Insulin Sensitization and Glucose Metabolism: Vaspin improves insulin sensitivity and glucose tolerance in both in vitro and in vivo models. Administration of recombinant vaspin to obese or insulin-resistant mice normalizes blood glucose, enhances insulin sensitivity, and modulates the expression of key adipocytokines (e.g., leptin, adiponectin, resistin, TNF-α).

• Obesity and Metabolic Syndrome Research: Vaspin acts as a compensatory adipokine in obesity, counteracting inflammation and metabolic dysfunction in adipose tissue and liver. Its levels are elevated in obesity and type 2 diabetes, making it a relevant biomarker and therapeutic target in these conditions.

• Cellular Mechanisms and Signaling Pathways: Recombinant vaspin activates key signaling pathways such as Akt and AMPK, which are central to metabolic regulation. It binds to the cell-surface GRP78/MTJ-1 complex, modulating endoplasmic reticulum (ER) stress responses and reducing ER stress-induced metabolic dysfunction.

• Cardiovascular and Anti-inflammatory Effects: Vaspin administration has demonstrated cardioprotective effects, improving cardiac structure and function in models of lipoatrophy-induced cardiomyopathy. It also reduces obesity-induced inflammation and endothelial cell apoptosis, suggesting broader roles in vascular and cardiac health.

• Experimental Applications: Recombinant human vaspin is used in:

  • Cell-based assays to study insulin signaling, glucose uptake, and inflammatory responses.
  • Animal models to investigate metabolic syndrome, obesity, diabetes, and cardiovascular disease.
  • Mechanistic studies of adipokine-receptor interactions and downstream signaling.

• Potential for Translational Research: Insights gained from recombinant vaspin studies may inform the development of new therapeutic strategies for metabolic and cardiovascular diseases, as vaspin is considered a promising target for intervention in obesity-related disorders.

In summary, recombinant human vaspin is a powerful tool for dissecting the molecular and physiological roles of this adipokine in metabolic health and disease, enabling both mechanistic and translational research across multiple systems.

Yes, recombinant human Vaspin can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common practice in commercial Vaspin ELISA kits, where recombinant human Vaspin is routinely used to generate standard curves for quantitative analysis.

Key considerations and supporting details:

• Commercial ELISA kits for Vaspin quantification use recombinant human Vaspin as the standard. These standards are typically provided in lyophilized form and reconstituted to prepare a dilution series for the standard curve.
• Purity and quantification: The recombinant protein used as a standard should be highly purified, and its concentration must be accurately determined, ideally by methods such as HPLC or absorbance at 280 nm with a known extinction coefficient.
• Parallelism: Studies and kit documentation indicate that standard curves generated with recombinant Vaspin are parallel to those obtained with natural Vaspin from biological samples, supporting its suitability for calibration.
• Matrix effects: When using recombinant Vaspin as a standard, ensure that the matrix (buffer or diluent) used for the standard curve matches the sample matrix as closely as possible to minimize matrix effects and ensure accurate quantification.
• Validation: If you are developing a custom ELISA or using a recombinant Vaspin standard not supplied with a kit, validate the standard curve for linearity, sensitivity, and parallelism with your sample type.

Best practices:

• Use freshly reconstituted or properly stored aliquots of recombinant Vaspin for each assay to avoid degradation or concentration changes.
• Prepare a serial dilution of the recombinant Vaspin to cover the expected concentration range in your samples.
• Confirm that your assay antibodies recognize both recombinant and native forms of Vaspin, as most commercial ELISAs are designed for this purpose.

In summary: Recombinant human Vaspin is widely accepted and validated as a standard for ELISA quantification, provided it is pure, accurately quantified, and validated for your specific assay conditions.

Recombinant Human Vaspin has been validated for several key applications in published research, primarily in the fields of metabolic disease, cell signaling, protease inhibition, and cancer biology.

Validated Applications:

• Cell-based assays:
  Recombinant human vaspin has been used to treat cultured cells (e.g., H-4-II-E-C3 hepatoma cells, endothelial cells, adipocytes, and cancer cell lines) to study its effects on signaling pathways, including phosphorylation of Akt and AMPK, and modulation of ER stress markers.

• Animal models:
  Administration of recombinant vaspin in mice (including diet-induced obesity and diabetic models) has been validated for improving glucose tolerance, insulin sensitivity, and reducing obesity-induced inflammation and metabolic dysfunction.

• Protease inhibition assays:
  Recombinant vaspin has been used in biochemical assays to demonstrate its serpin activity, specifically inhibiting human kallikrein 7 (hK7), and forming stable vaspin–enzyme complexes detectable by SDS-PAGE, Western blot, and mass spectrometry.

• Ex vivo tissue assays:
  Recombinant vaspin has been applied to isolated muscle and adipose tissue to assess its effects on insulin-stimulated glucose uptake.

• Cancer cell functional assays:
  Treatment of triple-negative breast cancer cell lines with recombinant vaspin has been validated for studying its effects on cell proliferation, migration, invasion, and microRNA expression (e.g., miR-33a-5p).

• Affinity purification and protein interaction studies:
  Recombinant vaspin has been used to identify and characterize its interaction with cell-surface receptors such as GRP78/MTJ-1 and GRP78/VDAC complexes, using affinity purification and knockdown experiments.

• Bioassays for adipokine function:
  Recombinant vaspin has been validated in bioassays to study its role as an adipokine, including effects on adiponectin expression and anti-inflammatory properties.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies, confirming recombinant human vaspin’s utility in mechanistic, functional, and translational research related to metabolism, inflammation, and cancer biology.

To reconstitute and prepare Recombinant Human Vaspin protein for cell culture experiments, dissolve the lyophilized protein in sterile 18 MΩ-cm H₂O at a concentration not less than 100 µg/mL. This stock solution can then be further diluted in appropriate sterile aqueous buffers compatible with your cell culture system.

Step-by-step protocol:

• Equilibrate: Allow the lyophilized vial and sterile water to reach room temperature before opening to minimize condensation.
• Centrifuge: Briefly centrifuge the vial to collect the powder at the bottom.
• Reconstitution: Add sterile 18 MΩ-cm H₂O to achieve at least 100 µg/mL. Gently mix by pipetting or slow vortexing; avoid vigorous shaking to prevent protein denaturation.
• Dissolution: Let the solution sit at room temperature for 15–30 minutes with gentle agitation. If undissolved particles remain, continue gentle mixing for up to 2 hours.
• Dilution: Dilute the stock solution to your desired working concentration using sterile cell culture medium or buffer. For cell culture, consider adding a carrier protein (e.g., 0.1% BSA) to minimize adsorption and loss, especially at low concentrations.
• Aliquoting and Storage: Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles. Store aliquots at 4°C for short-term use (2–7 days) or at −20°C to −80°C for long-term storage. Avoid freeze-thaw cycles to preserve protein integrity.

Additional considerations:

• Endotoxin: Confirm endotoxin levels are suitable for cell culture (<1 EU/µg is typical for sensitive applications).
• Sterility: Ensure all solutions and handling steps are performed aseptically to prevent contamination.
• Carrier protein: For long-term storage or low-concentration applications, add carrier protein (e.g., BSA at 0.1–1%) or glycerol (5–50%) to stabilize the protein.
• Compatibility: Before adding to cells, ensure the final buffer composition is compatible with your cell type and experimental design.

Summary Table:

This protocol ensures optimal solubility, stability, and biological activity of recombinant human Vaspin for cell culture experiments.