Recombinant Human FGF-23

Recombinant Human FGF-23 is widely used in research because it is a key regulator of phosphate and vitamin D metabolism, and its dysregulation is implicated in disorders such as chronic kidney disease, cardiovascular disease, and phosphate-wasting syndromes. Using recombinant FGF-23 allows precise, controlled studies of its biological effects, signaling pathways, and potential as a biomarker or therapeutic target.

Key reasons to use Recombinant Human FGF-23 in research applications:

• Phosphate and Vitamin D Homeostasis: FGF-23 is a hormone-like protein produced by osteocytes and osteoblasts that regulates phosphate excretion in the kidney and suppresses vitamin D activation. Recombinant FGF-23 enables mechanistic studies of these pathways in vitro and in vivo.

• Disease Modeling: Elevated FGF-23 levels are associated with chronic kidney disease (CKD), cardiovascular complications, and rare phosphate-wasting disorders. Recombinant protein is essential for modeling these conditions and testing interventions.

• Cellular and Molecular Mechanisms: Recombinant FGF-23 can be used to study its effects on various cell types, such as vascular smooth muscle cells, endothelial cells, and cardiomyocytes. For example, it has been shown to:

  • Induce proliferation and extracellular matrix remodeling in vascular cells.
  • Increase cardiomyocyte size and hypertrophic gene expression, implicating it in cardiac hypertrophy.
  • Modulate immune cell chemotaxis and erythroid differentiation.

• Signaling Pathways: FGF-23 acts via FGF receptors (primarily FGFR1 and FGFR3) and often requires the co-receptor α-Klotho for effective signaling. Recombinant protein is used to dissect these pathways, including downstream MAPK/ERK activation.

• Biomarker and Therapeutic Target Research: FGF-23 is a promising biomarker for cardiovascular and renal risk stratification and a potential therapeutic target in CKD and related disorders. Recombinant FGF-23 is used in assay development, validation, and therapeutic screening.

• Controlled Experimental Conditions: Recombinant proteins provide batch-to-batch consistency, defined activity, and species specificity, which are critical for reproducible and interpretable results in cell-based assays, animal models, and biochemical studies.

In summary, using recombinant human FGF-23 enables detailed, reproducible investigation of its physiological and pathological roles, supports the development of diagnostic and therapeutic strategies, and is essential for mechanistic studies in phosphate metabolism, cardiovascular biology, and kidney disease.

Yes, recombinant human FGF-23 can be used as a standard for quantification or calibration in ELISA assays, provided it matches the form of FGF-23 detected by your assay and is properly validated for this use. Many commercial ELISA kits for FGF-23 quantification use recombinant human FGF-23 as their calibrator or standard, and the assay is typically calibrated against this form.

Key considerations:

• Form of FGF-23: Ensure the recombinant protein is the same form (e.g., intact FGF-23, specific amino acid sequence) as the analyte your ELISA is designed to detect. Most intact FGF-23 ELISAs use recombinant human FGF-23 corresponding to amino acids 25–251 (Uniprot Q9GZV9) as the standard.
• Validation: The recombinant standard should be validated for parallelism and dilution linearity with endogenous FGF-23 in your sample matrix. This ensures that the recombinant protein behaves similarly to the native protein in the assay, avoiding matrix effects or non-parallel standard curves.
• Concentration Range: Prepare a standard curve using serial dilutions of the recombinant FGF-23 within the detection range of your ELISA (e.g., 0–1600 pg/mL or as specified by your kit).
• Documentation: Refer to your ELISA kit’s manual for recommended standard preparation and calibration procedures. Most kits provide detailed protocols for generating a standard curve using recombinant FGF-23.

Best practices:

• Use recombinant FGF-23 that is well-characterized and, if possible, sourced from a reputable supplier with documentation of purity and sequence.
• Confirm that the recombinant standard is compatible with your specific ELISA antibodies (capture and detection), as some assays are optimized for specific epitopes or forms of FGF-23.
• Validate the standard curve in your own laboratory conditions, especially if using a custom or in-house ELISA.

Summary Table: Use of Recombinant Human FGF-23 as ELISA Standard

In summary: Recombinant human FGF-23 is widely used as a standard for ELISA quantification, but it is essential to ensure form, validation, and compatibility with your assay for accurate calibration.

Recombinant Human FGF-23 has been validated for a range of applications in published research, primarily in studies of phosphate metabolism, mineral homeostasis, and related disease models. The most common validated applications include bioassays, cell culture, in vivo assays, ELISA standards, and functional assays.

Key validated applications:

• Bioassays: Used to study FGF-23’s biological activity, including its effects on phosphate regulation, parathyroid hormone suppression, and signaling pathways in various cell types (e.g., human, rat, mouse).
• Cell Culture: Applied to cultured cells to investigate FGF-23’s role in cellular processes such as erythroid differentiation, reticulocyte maturation, and vascular calcification.
• In Vivo Assays: Utilized in animal models (mouse, rat) to assess physiological and pathological effects, such as cardiac hypertrophy, fibrosis, and hypophosphatemia.
• ELISA Standard: Used as a standard protein for quantifying FGF-23 levels in biological samples via ELISA.
• Functional Assays: Employed to validate FGF-23’s activity, including its mitogenic and cell survival effects, and its ability to inhibit renal phosphate reabsorption.
• Protein Characterization: Validated for SDS-PAGE, mass spectrometry, and HPLC to confirm purity and identity.

Representative published research applications:

• Vascular Biology: Recombinant FGF-23 has been used to study its impact on vascular smooth muscle cell calcification and endothelial function, including effects mediated by reactive oxygen species.
• Renal and Parathyroid Function: Investigations into FGF-23’s suppression of parathyroid hormone and modulation of Klotho expression in kidney and parathyroid tissues.
• Disease Models: Extensively used in models of hypophosphatemic rickets, chronic kidney disease (CKD), and X-linked hypophosphatemia (XLH) to elucidate disease mechanisms and test therapeutic interventions.
• Immunoassays: Recombinant FGF-23 serves as a standard or control in immunoassays for quantifying endogenous FGF-23 in clinical and experimental samples.

Summary Table of Validated Applications

Additional Notes:

• Recombinant FGF-23 is not approved for clinical diagnostic use; its applications are limited to research settings.
• It is frequently used in studies investigating the molecular mechanisms of FGF-23 action and its therapeutic targeting, such as with monoclonal antibodies (e.g., burosumab).

These validated applications are supported by a broad body of published research, reflecting FGF-23’s central role in mineral metabolism and disease pathophysiology.

To reconstitute and prepare Recombinant Human FGF-23 protein for cell culture experiments, follow these general best practices, which are consistent across multiple suppliers and protocols:

Reconstitution

1. Centrifuge the vial briefly before opening to ensure all lyophilized powder is at the bottom.
2. Reconstitute the protein in sterile, endotoxin-free PBS (phosphate-buffered saline) or sterile distilled water. Some suppliers recommend adding carrier protein (e.g., 0.1% BSA) to stabilize the protein, especially if the protein is carrier-free.
   • Typical reconstitution concentration: 100 µg/mL (or as specified by the manufacturer).
   • For example:
     • Bio-Techne/R&D Systems: Reconstitute at 100 µg/mL in sterile PBS (with or without 0.1% BSA, depending on the product).
     • Abcam: Reconstitute in PBS.
     • Novus Biologicals: Reconstitute in sterile distilled water or aqueous buffer containing 0.1% BSA.
     • Cusabio: Reconstitute in deionized sterile water to 0.1–1.0 mg/mL.
3. Gently mix the solution by pipetting or swirling; avoid vigorous shaking or foaming.

Preparation for Cell Culture

1. Aliquot the reconstituted protein into small, single-use volumes to minimize freeze-thaw cycles.
2. Store aliquots at –20°C to –80°C for long-term storage. Avoid repeated freeze-thaw cycles.
3. For immediate use, dilute the reconstituted protein in cell culture medium (with or without serum, depending on your experimental design) to the desired working concentration.
4. Filter sterilize the diluted protein solution (0.22 µm filter) if necessary, especially for sensitive cell types.

Additional Notes

• Carrier protein (BSA): If your protein is carrier-free, adding 0.1% BSA can help stabilize it during storage and dilution.
• Glycerol: Some protocols recommend adding 5–50% glycerol (final concentration) to the reconstituted protein for improved stability during storage.
• Endotoxin levels: Ensure the protein is low in endotoxin (<0.005 EU/µg) for cell culture applications.

Example Protocol

1. Centrifuge the lyophilized FGF-23 vial.
2. Reconstitute in sterile PBS to 100 µg/mL (or as per manufacturer’s instructions).
3. Gently mix and aliquot into small volumes.
4. Store at –20°C to –80°C.
5. For cell culture, dilute to working concentration in culture medium and filter sterilize if needed.

Always refer to the specific product datasheet or certificate of analysis for detailed instructions and recommendations.