Recombinant Human FGF-21

Recombinant Human FGF-21 is widely used in research due to its potent regulatory effects on metabolism, its therapeutic potential in metabolic diseases, and its well-characterized activity in both in vitro and in vivo models.

Key reasons to use recombinant human FGF-21 in research applications include:

• Metabolic Regulation: FGF-21 is a hormone-like protein that plays a central role in regulating glucose and lipid metabolism. It activates key signaling pathways such as AMPK, SIRT1, and ERK, leading to improved insulin sensitivity, reduced blood glucose and lipid levels, and enhanced energy expenditure.

• Therapeutic Potential: Recombinant FGF-21 and its analogs have demonstrated efficacy in preclinical models for treating obesity, type 2 diabetes, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). These effects include reducing hepatic steatosis, inflammation, and fibrosis, as well as improving liver function and preventing further disease progression.

• Anti-inflammatory and Cytoprotective Effects: FGF-21 has been shown to attenuate inflammation and apoptosis in metabolic tissues, protect against liver injury, and modulate immune cell infiltration, making it valuable for studying mechanisms of tissue repair and metabolic disease pathogenesis.

• Biomarker and Diagnostic Utility: FGF-21 is being explored as a biomarker for metabolic disorders, including NAFLD and insulin resistance, due to its altered serum levels in these conditions.

• Experimental Versatility: Recombinant human FGF-21 is suitable for a range of applications, including:

  • In vitro studies of glucose uptake and adipocyte biology.
  • In vivo studies in rodent and primate models to assess metabolic, hepatic, and cardiovascular outcomes.
  • Mechanistic studies on signaling pathways involved in energy homeostasis and lipid metabolism.

• Improved Protein Quality: Recombinant production allows for high purity and batch-to-batch consistency, which is critical for reproducible experimental results.

• Emerging Clinical Relevance: FGF-21 analogs are under clinical investigation, and recombinant FGF-21 is partially used in clinical practice, supporting its translational relevance for drug development and biomarker discovery.

In summary, recombinant human FGF-21 is a valuable tool for investigating metabolic regulation, disease mechanisms, and therapeutic interventions in metabolic and inflammatory disorders, with robust evidence supporting its use in both basic and translational research.

Recombinant human FGF-21 can be used as a standard for quantification or calibration in ELISA assays, provided it is compatible with your assay system and matches the native protein recognized by your antibodies. Several commercial ELISA kits use recombinant human FGF-21 as their standard, and this is a common practice in quantitative immunoassays.

Key considerations and supporting details:

• Assay Compatibility: Many validated ELISA kits for FGF-21 quantification use recombinant human FGF-21 (often E. coli-expressed) as the standard, demonstrating that recombinant protein is suitable for generating standard curves. For example, one kit states: "It contains E. coli-expressed recombinant human FGF-21 and has been shown to accurately quantitate the recombinant factor". Another protocol specifies, "The recombinant protein is used as a Standard in this assay".

• Parallelism: It is important that the standard curve generated with recombinant FGF-21 is parallel to the response obtained with native FGF-21 in your samples. Some kits have validated that curves for recombinant and native FGF-21 are parallel, indicating equivalent immunoreactivity in the assay system.

• Protein Form: Ensure the recombinant FGF-21 you use matches the sequence and post-translational modifications (if any) of the native protein recognized by your ELISA antibodies. Most commercial standards are full-length, E. coli-expressed proteins, which may lack glycosylation but are generally accepted for calibration if the antibodies target linear epitopes.

• Reconstitution and Handling: Follow best practices for reconstitution and dilution of the recombinant standard, as described in ELISA kit protocols, to ensure accuracy and reproducibility.

• Documentation: If you are using a custom or non-kit recombinant FGF-21, document its source, sequence, and any modifications, and validate its performance in your specific ELISA system.

Summary Table: Use of Recombinant FGF-21 as ELISA Standard

In summary: You can use recombinant human FGF-21 as a standard for ELISA quantification, as long as it is compatible with your assay and validated for parallelism with native FGF-21. Always confirm compatibility with your specific ELISA system and document all relevant details for reproducibility.

Recombinant Human FGF-21 has been validated for a range of applications in published research, primarily in the context of metabolic regulation, wound healing, and cellular assays.

Key validated applications include:

• Metabolic Disease Models:
  Recombinant FGF-21 and its analogs have been used in preclinical and clinical studies for the treatment of nonalcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), type 2 diabetes, and severe hypertriglyceridemia. These studies demonstrate effects such as improved glucose and lipid metabolism, reduced hepatic fat accumulation, and improved liver histology in both animal models and human patients.

• Cellular Bioassays:
  FGF-21 has been validated in cell proliferation assays (e.g., BaF3 mouse pro-B cell line transfected with human FGFRIIIc), adipocyte differentiation assays, and signaling pathway activation (e.g., phosphorylation of Erk1/2 in 3T3-L1 adipocytes).

• Wound Healing and Angiogenesis:
  Recombinant FGF-21 has been shown to promote wound healing in diabetic models by restoring endothelial cell proliferation, migration, tube formation, and reducing inflammation under hyperglycemic conditions. Both in vitro and in vivo studies confirm its role in stimulating angiogenesis and anti-inflammatory responses, with efficacy comparable to VEGF in diabetic wound models.

• In Vivo Animal Studies:
  Applications include in vivo efficacy studies in mouse models of obesity, diabetes, and liver disease, where FGF-21 administration leads to improved metabolic parameters and tissue histology.

• ELISA and Immunoassays:
  Recombinant FGF-21 has been used as a capture antigen in ELISA for quantifying FGF-21 levels in biological samples.

• Western Blot and Molecular Biology:
  It is used as a positive control or for stimulating signaling pathways in Western blot and qRT-PCR analyses to assess downstream gene expression and protein phosphorylation.

• Transdermal Delivery and Fusion Protein Studies:
  Novel recombinant FGF-21 constructs, including those fused with cell-penetrating peptides, have been validated for transdermal drug delivery systems (TDDS) and show enhanced skin penetration and pharmacological activity in wound healing models.

• Biomarker Research:
  Circulating FGF-21 levels are investigated as biomarkers for metabolic diseases, including diabetes progression and diabetic kidney disease.

Summary Table of Validated Applications

These applications are supported by both mechanistic studies and translational research, highlighting the versatility of recombinant human FGF-21 in metabolic, cellular, and regenerative medicine contexts.

To reconstitute and prepare Recombinant Human FGF-21 protein for cell culture experiments, add sterile water or sterile PBS to the lyophilized protein to achieve a concentration of 0.1–1.0 mg/mL, with 100 μg/mL being a common working concentration. For optimal stability and activity, include a carrier protein such as 0.1–1% BSA or HSA in your buffer.

Step-by-step protocol:

• Centrifuge the vial briefly to collect all lyophilized material at the bottom before opening.
• Add sterile water or PBS:
  • For most applications, reconstitute at 100 μg/mL in sterile PBS or sterile water.
  • If using PBS, ensure pH is 7.2–7.4.
• Add carrier protein:
  • For cell culture, add 0.1–1% BSA or HSA to minimize adsorption and stabilize the protein.
• Mix gently: Allow the solution to sit at room temperature for several minutes, then gently vortex or pipette to fully dissolve the protein.
• Aliquot: Divide into single-use aliquots to avoid repeated freeze-thaw cycles.
• Storage:
  • Short-term: 2–8°C for up to 1 week.
  • Long-term: –20°C to –80°C for several months.
• Avoid repeated freeze-thaw cycles to preserve bioactivity.

Dilution for cell culture:

• Dilute the reconstituted stock to your desired working concentration in cell culture medium immediately before use.
• If possible, filter-sterilize the final working solution using a 0.2 μm filter.

Notes:

• Always consult the specific product datasheet for any unique formulation or reconstitution instructions, as excipients and recommended buffers may vary.
• For bioassays, ensure the protein is carrier-free if required, as BSA or HSA may interfere with certain applications.

Summary of key points:

• Reconstitute at 100 μg/mL in sterile PBS or water, with 0.1–1% BSA/HSA.
• Aliquot and store at –20°C to –80°C.
• Avoid repeated freeze-thaw cycles.
• Dilute to working concentration in cell culture medium just before use.

These steps will ensure that recombinant FGF-21 is optimally prepared for reliable and reproducible cell culture experiments.