Recombinant Human Visfatin

Using Recombinant Human Visfatin in research applications is valuable because visfatin (also known as NAMPT or PBEF) is a multifunctional adipocytokine with critical roles in metabolism, inflammation, cell survival, and cardiovascular function. Recombinant forms enable controlled, reproducible studies of its biological effects in vitro and in vivo.

Key scientific reasons to use recombinant human visfatin include:

• Metabolic Regulation: Visfatin exhibits insulin-mimetic effects, enhancing insulin sensitivity, promoting glucose uptake, and lowering plasma glucose levels in cell and animal models. This makes it a useful tool for studying diabetes, obesity, and metabolic syndrome.

• Inflammation and Immunomodulation: Visfatin acts as a pro-inflammatory cytokine, modulating immune cell activation and cytokine production (e.g., IL-6, IL-8, MCP-1), and is involved in key inflammatory signaling pathways such as NF-κB and MAPK. It is widely used to model and dissect inflammatory responses in immune and non-immune cells.

• Cell Survival, Apoptosis, and Proliferation: Recombinant visfatin influences cell proliferation, apoptosis, and necrosis, often via mitochondrial and STAT3 signaling pathways. It can be used to study mechanisms of cell death and survival in various cell types, including cardiac and immune cells.

• Cardiovascular Research: Visfatin is implicated in endothelial dysfunction, atherosclerosis, vascular remodeling, and myocardial protection against ischemia-reperfusion injury. Recombinant visfatin is used to investigate these processes and potential therapeutic interventions.

• Angiogenesis and Tissue Remodeling: It upregulates VEGF and matrix metalloproteinases (MMPs), promoting angiogenesis and tissue remodeling, which is relevant for cancer, wound healing, and vascular biology studies.

• Molecular Mechanisms: As the rate-limiting enzyme in NAD+ biosynthesis, visfatin/NAMPT is central to cellular energy metabolism, sirtuin activation, DNA repair, and stress responses. Recombinant protein allows precise manipulation of these pathways in experimental systems.

• Standardization and Reproducibility: Recombinant human visfatin provides a consistent, defined reagent for dose-response studies, mechanistic assays, and biomarker validation, avoiding variability inherent to tissue extracts or serum samples.

In summary, recombinant human visfatin is a versatile tool for dissecting its diverse biological functions in metabolism, inflammation, cardiovascular health, and cell biology, supporting both basic research and translational studies.

Yes, recombinant human Visfatin can be used as a standard for quantification or calibration in ELISA assays, provided it is compatible with the antibodies and detection system of your specific assay. Most commercial human Visfatin ELISA kits are designed to quantify both natural and recombinant forms of Visfatin, and their standard curves are typically generated using recombinant human Visfatin.

Key considerations:

• Assay Compatibility: Ensure that the recombinant Visfatin you use as a standard is recognized by the capture and detection antibodies in your ELISA. Most kits specify that they detect both natural and recombinant human Visfatin.
• Standard Curve Preparation: Prepare the standard curve using serial dilutions of recombinant Visfatin in the same buffer or matrix as your samples to minimize matrix effects.
• Validation: Confirm that the recombinant standard yields a linear and reproducible standard curve within the assay’s detection range (e.g., 0.16–10 ng/mL, 0.125–8 ng/mL, or as specified by your kit).
• Recovery and Precision: Check that recovery rates and intra/inter-assay precision meet acceptable criteria when using recombinant Visfatin as a standard.

Scientific best practices:

• Use recombinant Visfatin with a verified sequence and purity, ideally referenced to UniProt ID P43490, to ensure consistency.
• Validate the standard curve with known concentrations and, if possible, compare with kit-provided standards to confirm equivalence.
• Document any deviations from the kit protocol and include appropriate controls.

Summary Table: Recombinant Human Visfatin as ELISA Standard

In conclusion, recombinant human Visfatin is widely accepted as a standard for ELISA quantification, but assay-specific validation is essential for accurate calibration.

Recombinant Human Visfatin has been validated in a range of published research applications, primarily in cell-based bioassays, in vivo studies, and mechanistic signaling investigations related to inflammation, metabolism, angiogenesis, and cancer biology.

Key validated applications include:

• Cell-based bioassays:

  • Induction of VEGF secretion in PC-3 human prostate cancer cells, with a reported ED₅₀ of 1–5 μg/mL.
  • Stimulation of VEGF-C expression and lymphangiogenesis in esophageal squamous cell carcinoma (ESCC) and other cancer cell lines, often assessed by qPCR, ELISA, and Western blot.
  • Promotion of inflammatory cytokine production in various immune cell types, often measured by ELISA or multiplex cytokine assays.
  • Enhancement of cell proliferation and upregulation of genes such as hTERT in cancer cell models.

• In vivo studies:

  • Regulation of feeding behavior in goldfish, demonstrating physiological effects in animal models.
  • Modulation of fertility and oocyte development in murine models.

• Mechanistic and signaling pathway studies:

  • Activation of NF-κB, MEK/ERK, and MAPK signaling pathways, often validated by luciferase reporter assays, Western blot, and pharmacological inhibition.
  • Investigation of insulin-mimetic effects and roles in glucose homeostasis in metabolic disease models.

• Angiogenesis and vascular biology:

  • Promotion of angiogenesis in vitro and in vivo, including upregulation of VEGF and matrix metalloproteinases (MMP-2, MMP-9) in endothelial cells.

• Immunomodulation:

  • Studies on T and B cell development and immunosuppressive effects in tumor microenvironments, including modulation of PD-1 expression on CD8+ T cells.

• Protein standards and controls:

  • Use as a standard in ELISA and other immunoassays for quantification and validation purposes.

Summary Table of Validated Applications

Additional notes:

• Most studies use recombinant human visfatin in concentrations ranging from nanograms to micrograms per milliliter, depending on the assay and cell type.
• Applications span both basic mechanistic research and disease models, particularly in cancer, metabolic, and inflammatory contexts.
• Validation methods include ELISA, Western blot, qPCR, luciferase reporter assays, and in vivo physiological measurements.

If you require protocol details or specific assay conditions for any of these applications, please specify the context or experimental system of interest.

To reconstitute and prepare Recombinant Human Visfatin protein for cell culture experiments, follow these general best practices, which are consistent across multiple suppliers and protocols:

Reconstitution Steps

1. Centrifuge the Vial: Before opening, briefly centrifuge the lyophilized protein vial to ensure all powder is at the bottom.

2. Reconstitution Solution: Most recombinant human Visfatin proteins are reconstituted in sterile PBS (phosphate-buffered saline) or sterile distilled water. For example:

   • R&D Systems recommends reconstituting at 500 µg/mL in sterile PBS.
   • Bio-Rad suggests reconstituting with 0.25 mL distilled water.
   • Enzo Biochem recommends sterile 20 mM HCl at 0.1 mg/mL.

   Always check the specific product datasheet for the recommended buffer.

3. Gentle Mixing: Gently pipette the solution down the sides of the vial to avoid foaming. Allow the vial to sit at room temperature for 15–30 minutes with gentle agitation to fully dissolve the protein. Avoid vigorous shaking.

4. Storage After Reconstitution:

   • For short-term use (2–7 days), store the reconstituted protein at 4°C.
   • For long-term storage, aliquot the protein and store at –20°C or –80°C. Avoid repeated freeze-thaw cycles.

Preparation for Cell Culture

• Dilution: Dilute the reconstituted protein in cell culture medium or an appropriate buffer to the desired working concentration. For example, R&D Systems reports an ED50 of 1–5 µg/mL for VEGF secretion in PC-3 cells.
• Carrier Protein: If the protein is carrier-free, consider adding a carrier protein (e.g., 0.1% BSA) for long-term storage to prevent adsorption to surfaces.

Additional Tips

• Always use sterile techniques to prevent contamination.
• Confirm the endotoxin level of the protein if sensitive cell types are used.
• Optimize the concentration for your specific cell type and assay.

By following these steps, you can ensure the proper reconstitution and preparation of Recombinant Human Visfatin protein for cell culture experiments.