Recombinant Human IGF-II/IGF2 Protein

Recombinant Human IGF-II/IGF2 Protein is widely used in research due to its critical roles in cell proliferation, differentiation, neuroprotection, angiogenesis, and disease modeling, making it a versatile tool for both basic and translational studies.

Key scientific applications and rationale include:

• Cell Proliferation and Differentiation: IGF-II stimulates proliferation in various cell lines, such as MCF-7 human breast cancer cells, and supports embryonic development and energy metabolism. This makes it valuable for studies on cancer biology, developmental biology, and tissue regeneration.

• Neuroprotection and Cognitive Function: IGF-II has demonstrated neurotrophic effects, including stimulation of neurogenesis, synaptogenesis, and protection against neurotoxicity in rodent models of neurodegenerative diseases like Alzheimer’s disease. Its use enables investigation into mechanisms of memory formation, neurodegeneration, and potential therapeutic interventions.

• Angiogenesis and Vascular Biology: IGF-II and its variants promote endothelial cell migration, tube formation, and blood vessel formation, supporting research in angiogenesis, wound healing, and vascular remodeling.

• Immunomodulation: IGF-II enhances regulatory T-cell functions and can suppress allergic responses, providing a tool for immunology and allergy research.

• Disease Modeling and Therapeutic Development: Recombinant IGF-II is used in bioassays to study tumor growth, metastasis, and fibrotic processes, as well as in models of fetal growth restriction and metabolic disorders. It also facilitates the development and testing of novel therapeutics, such as enzyme fusion proteins for improved cellular uptake.

• Mechanistic Studies: IGF-II interacts with multiple receptors (IGF1R, IGF2R, IR) and binding proteins, allowing detailed analysis of signaling pathways, receptor specificity, and protein-protein interactions.

• Assay Development: Recombinant IGF-II is suitable for ELISA capture, binding assays, and in vivo studies, providing standardized reagents for quantitative and qualitative analyses.

In summary, using recombinant human IGF-II/IGF2 protein enables precise control over experimental conditions, reproducibility, and mechanistic insight into diverse biological processes relevant to development, disease, and therapeutic innovation.

Yes, recombinant human IGF-II/IGF2 protein can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately known. This practice is common in research ELISAs for IGF2, and several commercial ELISA kits use recombinant IGF2 as their calibrator or standard.

Key considerations and supporting details:

• Recombinant IGF2 as Standard: Many validated ELISA kits for human IGF2 quantification use recombinant human IGF2, typically expressed in E. coli, as their standard for generating calibration curves. These standards are used to interpolate sample concentrations based on the assay’s standard curve.

• Parallelism and Linearity: Studies and kit documentation indicate that standard curves generated with recombinant IGF2 are parallel to those generated with natural IGF2, supporting the validity of using recombinant protein for quantification. This ensures that results obtained with recombinant standards are comparable to those with endogenous IGF2.

• Calibration Against International Standards: Some ELISA kits calibrate their recombinant IGF2 standards against international reference reagents, such as the WHO NIBSC 96/538 standard, to ensure traceability and comparability of results. If your application requires absolute quantification in international units, calibrating your recombinant standard against such a reference is recommended.

• Carrier Protein Considerations: Recombinant IGF2 is available in carrier-free and carrier-added (e.g., BSA) forms. Carrier-free protein is preferred if BSA or other additives could interfere with your assay, while carrier-added forms may offer greater stability for repeated use.

• Documentation and Validation: Ensure that the recombinant IGF2 you use is well-characterized (purity, sequence, activity) and that you prepare the standard curve in the same matrix as your samples (e.g., serum, plasma, buffer) to minimize matrix effects.

• Best Practices:

  • Prepare a fresh standard curve for each assay run.
  • Validate the linearity and parallelism of your standard curve with your sample matrix.
  • If using a new lot or source of recombinant IGF2, verify its performance in your assay system.

Summary Table: Use of Recombinant IGF2 as ELISA Standard

In summary, recombinant human IGF-II/IGF2 protein is suitable and widely used as a standard for ELISA quantification, provided you follow best practices for calibration and validation in your specific assay context.

Recombinant Human IGF-II/IGF2 Protein has been validated for a wide range of applications in published research, primarily in the context of cell signaling, proliferation, differentiation, and disease modeling. Key validated applications include:

• Bioassays/Functional Assays: Used to assess biological activity, such as stimulation of cell proliferation (e.g., MCF-7 breast cancer cells), migration, differentiation, and survival in various cell types.
• ELISA (Enzyme-Linked Immunosorbent Assay): Employed as a standard or capture antigen for quantifying IGF2 in biological samples, including plasma and cell culture supernatants.
• Western Blot: Used as a positive control or for antibody validation in protein detection assays.
• Blocking Assays: Applied to investigate the specificity of IGF2-mediated signaling pathways by competitive inhibition.
• Immunohistochemistry: Utilized for tissue localization studies of IGF2 or its receptors.
• Binding Assays: Used to characterize interactions with IGF2 receptors or binding proteins.
• In Vivo Studies: Administered to animal models (rodents, chicken) to study effects on memory, neuroprotection, neurogenesis, and disease phenotypes such as Alzheimer’s disease, neurodevelopmental disorders, and cancer.
• Cellular Uptake and Delivery Studies: Used as a fusion partner (e.g., rhNAGLU-IGF-II) to enhance cellular uptake of therapeutic proteins via the IGF2 receptor pathway.

Representative published research applications:

• Cancer research: IGF2 has been used in bioassays to study its role in cancer cell proliferation, migration, invasion, and drug resistance, particularly in breast cancer and sarcoma models.
• Neuroscience: Exogenous IGF2 administration in rodent models has been shown to enhance memory, promote neurogenesis, and provide neuroprotection in models of Alzheimer’s disease and other CNS disorders.
• Stem cell and developmental biology: IGF2 is used to promote cell growth, differentiation, and survival in stem cell cultures and during studies of muscle and bone development.
• Immunology: IGF2 has been shown to modulate regulatory T-cell function and suppress allergic responses in experimental models.
• Metabolic and endocrine research: Used to study insulin secretion, glucose metabolism, and osteogenic activity.

Summary Table of Validated Applications

These applications are supported by both product validation data and peer-reviewed publications, demonstrating the versatility of recombinant human IGF-II/IGF2 protein in basic and translational research.

To reconstitute and prepare Recombinant Human IGF-II/IGF2 protein for cell culture experiments, follow these best-practice steps based on multiple authoritative protocols:

1. Centrifuge the vial before opening to ensure all lyophilized protein is at the bottom.

2. Reconstitution:

   • Use sterile, endotoxin-free water or sterile PBS (pH 7.2–7.4) as the solvent. The specific diluent may depend on the manufacturer’s recommendation, but both are commonly used for IGF-II.
   • Add solvent to achieve a stock concentration of 100–200 μg/mL. For example:
     • Some protocols recommend not less than 200 μg/mL in sterile water.
     • Others suggest 100 μg/mL in sterile PBS.
   • Gently mix (do not vortex) and incubate at room temperature for at least 20 minutes to ensure complete dissolution.

3. Carrier Protein (Optional but Recommended for Stability):

   • If the protein will be stored or diluted to low concentrations, add a carrier protein such as 0.1% recombinant human serum albumin (HSA) or 0.1% BSA to minimize adsorption and loss.
   • This is especially important for working concentrations below 10 μg/mL.

4. Aliquoting and Storage:

   • Prepare single-use aliquots to avoid repeated freeze-thaw cycles.
   • Store aliquots at –20 °C to –80 °C for long-term storage, or at 2–8 °C for up to one month after reconstitution.
   • Avoid repeated freeze-thaw cycles, as this can reduce protein activity.

5. Working Solution Preparation:

   • Dilute the stock solution into your cell culture medium immediately before use.
   • Typical working concentrations for cell culture applications range from 1–100 ng/mL, depending on the cell type and experimental design.

Summary Table: IGF-II Reconstitution and Preparation

Additional Notes:

• Always consult the specific product datasheet for any unique instructions, as formulations may vary.
• If the protein is lyophilized from acetic acid or TFA, ensure complete removal or neutralization before use in sensitive cell types.
• Use aseptic technique throughout to maintain sterility.

These steps will ensure optimal solubility, stability, and bioactivity of recombinant human IGF-II/IGF2 for cell culture experiments.