Recombinant Mouse IGFBP-3

Recombinant mouse IGFBP-3 offers several compelling advantages for research applications across multiple biological systems and experimental contexts.

Developmental Biology Applications

Recombinant mouse IGFBP-3 enhances preimplantation embryo development in culture systems. When added to embryo culture media, it accelerates developmental progression through early cell divisions, increasing the rate of advancement to the 2-cell, 4-cell, 5-cell, and 8-cell stages compared to control conditions. This improvement occurs through activation of critical signaling pathways, including phosphorylation of Akt and ribosomal protein S6, which promote cell proliferation and survival. This makes it particularly valuable for embryo culture optimization and reproductive research.

Signaling Pathway Versatility

The protein activates multiple interconnected signaling cascades that extend beyond simple growth factor sequestration. IGFBP-3 transactivates both IGF1 and EGF receptors through a shared downstream pathway, activating the PI3-kinase/Akt signaling cascade. Additionally, it can interact with TGFβ receptors to activate Smad signaling pathways and regulate cell proliferation. The protein also functions intracellularly, translocating into the nucleus and cytoplasm where it regulates gene transcription, DNA repair, and interacts with nuclear hormone receptors.

Therapeutic and Disease Model Applications

Recombinant mouse IGFBP-3 demonstrates therapeutic potential in inflammatory disease models. It inhibits allergic lung inflammation, reduces vascular endothelial growth factor (VEGF) production, and decreases vascular leak in asthma models. In cancer research, peptide derivatives from IGFBP-3's terminal domain show promise for targeted cancer cell therapy, with demonstrated efficacy in reducing tumor burden in xenograft models.

Cell Migration and Tumor Biology

The protein plays important roles in cell migration independent of cell cycle progression, particularly in cell lines with high metastatic potential. This makes it valuable for studying tumor cell behavior and metastatic mechanisms in various cancer models.

Experimental Advantages

Recombinant mouse IGFBP-3 provides a standardized, reproducible reagent for investigating endogenous IGFBP-3 functions. Since the protein is naturally secreted by endometrial and oviductal cells, exogenous recombinant forms allow controlled experimental manipulation of physiological concentrations. The protein's ability to regulate multiple biological processes—including gene transcription, DNA damage responses, apoptosis, and autophagy—through interactions with non-IGF-related ligands makes it a versatile tool for mechanistic studies.

Yes, recombinant mouse IGFBP-3 can be used as a standard for quantification and calibration in ELISA assays. In fact, recombinant mouse IGFBP-3 is the standard product specifically formulated for this purpose in commercial ELISA kits.

Standard Preparation and Use

Recombinant mouse IGFBP-3 standards are typically supplied in lyophilized form and require reconstitution before use. The protein is reconstituted in the provided sample diluent buffer, and serial dilutions are prepared to establish a standard curve across the assay's measurement range. For example, standard curves commonly span from approximately 156 pg/mL to 5000 pg/mL, depending on the specific assay configuration.

Quality Specifications

The recombinant protein used in ELISA kits typically meets rigorous quality standards, with purity levels of 95% or higher as determined by SDS-PAGE analysis. The molecular weight of recombinant mouse IGFBP-3 is approximately 29.4 kDa. These specifications ensure consistent and reliable quantification across multiple assay runs.

Practical Considerations

When using recombinant mouse IGFBP-3 as a standard, the protein concentration of your samples is interpolated directly from the standard curve generated during the assay. For optimal results, recombinant protein formulated with stabilizing agents such as bovine serum albumin (BSA) is recommended for use in cell or tissue culture applications and as ELISA standards. This formulation helps maintain protein stability and prevents non-specific binding during the assay.

The sandwich ELISA format recognizes both natural and recombinant forms of mouse IGFBP-3, making the recombinant standard suitable for quantifying endogenous protein in your biological samples.

Recombinant Mouse IGFBP-3 has been validated for several key applications in published research, primarily in the following areas:

• Bioactivity assays: Recombinant Mouse IGFBP-3 is routinely validated for its ability to modulate IGF (insulin-like growth factor) activity, such as inhibiting IGF-II-induced proliferation in cell lines like MCF-7. This confirms its functional integrity and relevance for mechanistic studies.

• Cell signaling and functional studies: It has been used to investigate signaling pathways in various cell types, including:

  • Inhibition of melanoma cell migration, invasion, and tumor growth in both human and murine melanoma models, with effects shown to be independent of IGF-1 and mediated via the Akt-GSK3β pathway.
  • Acceleration of preimplantation embryo development in mice, with demonstrated activation of Akt and rpS6 phosphorylation, and interactions with IGF1R and EGFR signaling pathways.
  • Modulation of cell proliferation, gene transcription, and DNA repair through nuclear and cytoplasmic interactions.

• ELISA and immunoassays: Recombinant Mouse IGFBP-3 is validated as a standard or control in quantitative ELISA assays for measuring IGFBP-3 levels in mouse serum, plasma, or cell culture supernatants. These assays confirm specificity, sensitivity, and reproducibility for detection and quantification purposes.

• Western blotting: It is used as a positive control or standard in Western blot analyses to confirm antibody specificity and protein detection.

• In vivo studies: Recombinant Mouse IGFBP-3 has been administered to mice to study its effects on tumor growth, embryo development, and other physiological processes, validating its bioactivity and relevance in animal models.

• Mechanistic studies: Research has used recombinant IGFBP-3 to dissect its role in cell adhesion, apoptosis, inflammation, and interaction with nuclear and membrane receptors, including studies on gene transcription and DNA repair.

Summary of validated applications:

• Bioactivity/cell proliferation assays
• Cell signaling pathway analysis
• ELISA (quantitative immunoassays)
• Western blotting
• In vivo functional studies (e.g., tumor inhibition, embryo development)
• Mechanistic studies of protein-protein and protein-DNA interactions

These applications are supported by both product validation data and peer-reviewed research, confirming the utility of recombinant Mouse IGFBP-3 in diverse experimental contexts.

To reconstitute and prepare Recombinant Mouse IGFBP-3 protein for cell culture experiments, dissolve the lyophilized protein in sterile buffer—commonly sterile PBS or distilled water—to achieve the desired concentration, typically between 0.1–0.5 mg/mL for stock solutions. Avoid vortexing to prevent protein denaturation.

Step-by-step protocol:

• Reconstitution:

  • Add sterile PBS or distilled water directly to the lyophilized IGFBP-3 protein.
  • Target a stock concentration such as 100 μg/mL (as used in published mouse embryo studies) or up to 0.5 mg/mL depending on your experimental needs.
  • Gently mix by pipetting or slow inversion; do not vortex.
  • Allow the protein to fully dissolve at room temperature for several minutes.

• Aliquoting and Storage:

  • Once reconstituted, aliquot the solution to avoid repeated freeze-thaw cycles.
  • Store aliquots at 2–8°C for up to one month, or at –20°C to –70°C for longer-term storage.
  • For short-term use (days), refrigeration at 4°C is acceptable.

• Preparation for Cell Culture:

  • Dilute the stock solution into your cell culture medium to the final working concentration immediately before use.
  • Filter-sterilize if necessary, especially if the buffer or water was not sterile.
  • Typical working concentrations for functional assays range from 10–100 ng/mL, but optimal dosing should be determined empirically for your cell type and assay.

Additional notes:

• Avoid repeated freeze-thaw cycles to maintain protein integrity.
• If using for sensitive assays, consider adding carrier protein (e.g., 0.1% BSA) to minimize adsorption losses, unless this interferes with your experiment.
• Always consult the specific product datasheet for any manufacturer-recommended buffer or concentration, as some recombinant proteins may require special handling.

Summary Table:

This protocol ensures protein stability and bioactivity for cell culture applications. Adjust concentrations and buffers as needed for your specific experimental design.