Recombinant Human IGFBP-4

Recombinant Human IGFBP-4 is used in research applications to study its regulatory effects on insulin-like growth factor (IGF) signaling, bone metabolism, cancer biology, cellular senescence, and angiogenesis. Its recombinant form ensures high purity, batch-to-batch consistency, and suitability for mechanistic studies in vitro and in vivo.

Key scientific applications and rationales include:

• IGF Modulation: IGFBP-4 binds IGF-I and IGF-II with high affinity, modulating their bioavailability and activity. In bone research, recombinant IGFBP-4 can inhibit IGF-I-induced bone formation when administered locally, making it a valuable tool for dissecting IGF-dependent pathways in osteogenesis and bone remodeling.

• Cancer Research: IGFBP-4 exhibits context-dependent effects on cancer cell growth, motility, and invasion. It can act as an inhibitor or promoter depending on the cancer type and microenvironment. Recombinant IGFBP-4 is used to study these mechanisms and to evaluate its potential as a therapeutic or biomarker in oncology.

• Anti-Angiogenic and Anti-Tumorigenic Effects: Recombinant IGFBP-4, particularly its C-terminal fragment, has demonstrated potent anti-angiogenic and anti-tumorigenic properties independent of IGF binding. These effects are relevant for research into tumor biology and the development of anti-cancer strategies.

• Cellular Senescence and Aging: Systemic administration of recombinant IGFBP-4 in animal models increases cellular senescence and reduces stemness in mesenchymal stem cells, implicating it in aging and tissue regeneration studies.

• Cardiovascular and Developmental Biology: IGFBP-4 modulates Wnt signaling and supports cardiomyocyte differentiation, making it useful for studies in cardiac development and regenerative medicine.

• Biomarker and Diagnostic Research: Recombinant IGFBP-4 is used as a standard in immunoassays (e.g., ELISA, Western blot) to quantify endogenous IGFBP-4 in biological samples, supporting biomarker discovery in various diseases.

• Reproducibility and Consistency: The recombinant form allows for controlled experimental conditions, eliminating variability associated with native protein purification and enabling precise dose-response and mechanistic studies.

In summary, using recombinant human IGFBP-4 enables precise investigation of IGF signaling, bone and cancer biology, angiogenesis, aging, and biomarker development, providing a versatile tool for both basic and translational research.

Yes, recombinant human IGFBP-4 can generally be used as a standard for quantification or calibration in ELISA assays, provided the assay is validated to recognize both recombinant and native forms of IGFBP-4. This is a common practice in quantitative ELISA protocols, but several technical considerations must be addressed to ensure accuracy.

Key considerations:

• Assay Specificity: Some ELISA kits are designed to detect only native IGFBP-4 and may not recognize recombinant forms due to differences in folding, glycosylation, or other post-translational modifications. However, many commercial and custom ELISA kits are validated to detect both native and recombinant IGFBP-4. Always check the kit documentation or validate the assay with your recombinant standard.

• Calibration and Standard Curve: Recombinant IGFBP-4 is frequently used to generate standard curves for quantitative ELISA, allowing for accurate determination of IGFBP-4 concentrations in biological samples. The standard should be highly purified and its concentration accurately determined.

• Matrix Effects: The diluent used for the standard (often a buffer) may differ from the sample matrix (e.g., serum, plasma, cell culture supernatant), which can affect assay performance. Internal controls and matrix-matched standards are recommended to account for potential differences in detection.

• Validation: If using recombinant IGFBP-4 as a standard, validate that the assay yields comparable results for both recombinant and native IGFBP-4. This can be done by spiking known amounts of recombinant protein into sample matrices and confirming recovery and linearity.

Best Practices:

• Use a recombinant IGFBP-4 standard that is as similar as possible to the native protein in terms of structure and purity.
• Prepare a full calibration curve with multiple concentrations of the recombinant standard for each assay run.
• Include appropriate controls to ensure assay accuracy and reproducibility.
• Consult the ELISA kit protocol to confirm compatibility with recombinant standards and follow recommended procedures for standard preparation.

Summary Table: Recombinant IGFBP-4 as ELISA Standard

In conclusion: Recombinant human IGFBP-4 is suitable as a standard for ELISA quantification if the assay is validated for both forms. Always confirm assay compatibility and validate performance with your specific recombinant preparation.

Research Applications of Recombinant Human IGFBP-4

Recombinant human IGFBP-4 (rhIGFBP-4) has been validated across multiple research applications in published literature:

Immunoassay Development and Validation

Recombinant IGFBP-4 has been extensively used as an antigen, tracer, and standard in radioimmunoassay (RIA) development. The protein was expressed in Escherichia coli as a glutathione S-transferase fusion protein and demonstrated equivalent binding properties, electrophoretic migration, and immunoreactivity to native IGFBP-4 purified from biological sources. This validation enabled sensitive measurement of circulating IGFBP-4 levels in human serum, with recovery rates exceeding 90%.

Cell Proliferation and Bioassay Studies

The biological activity of recombinant IGFBP-4 has been established through cell proliferation assays using the alamarBlue assay on MG63 osteosarcoma cells. These studies demonstrated that intact recombinant IGFBP-4 potently inhibits IGF-I and IGF-II-induced cell proliferation, requiring only 40 ng/ml to block 50% of the IGF effect, compared to 2560 ng/ml for proteolytic IGFBP-4 fragments. The protein has also been validated in bioassays examining oocyte maturation and embryo development in follicular fluid studies.

In Vivo Bone Formation Studies

Recombinant IGFBP-4 has been validated in murine models examining its effects on bone formation parameters. Local administration completely blocked IGF-I-induced increases in alkaline phosphatase activity in parietal bone, while systemic administration increased bone formation markers in both serum and bone extracts. These studies established that recombinant IGFBP-4 exhibits differential effects depending on route of administration.

Biomarker and Diagnostic Applications

IGFBP-4 proteolytic fragments derived from recombinant protein have been validated as biomarkers for predicting major adverse cardiovascular events (MACE) in acute coronary syndrome patients. Additionally, IGFBP-4 has been validated in diagnostic contexts for solid tumors, including nasopharyngeal carcinoma and ovarian cancer.

Analytical Techniques

Recombinant IGFBP-4 has been validated for use in Western blotting, HPLC, SDS-PAGE, and ELISA applications.

To reconstitute and prepare Recombinant Human IGFBP-4 protein for cell culture experiments, follow these steps for optimal protein stability and biological activity:

1. Centrifuge the vial briefly before opening to ensure all lyophilized powder is at the bottom.
2. Reconstitute the protein in sterile PBS (phosphate-buffered saline) or sterile distilled water. The recommended concentration range is 0.1–1.0 mg/mL for stock solutions.
3. Gently mix the solution by swirling or gentle pipetting. Avoid vortexing or vigorous pipetting to prevent protein denaturation.
4. For long-term storage, add a carrier protein or stabilizer such as 0.1% BSA (bovine serum albumin), 5% HSA (human serum albumin), 10% FBS (fetal bovine serum), or 5% trehalose. This helps maintain protein stability during freezing.
5. Aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles, which can degrade the protein.
6. Store aliquots at -20°C or lower (preferably -70°C for extended storage). Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
7. For cell culture experiments, dilute the stock solution to the desired working concentration using cell culture medium or PBS containing carrier protein (e.g., PBS + 0.1% BSA).

Summary Table: Reconstitution Protocol

Additional Notes:

• Always consult the specific product datasheet or Certificate of Analysis for any manufacturer-specific instructions regarding buffer composition or concentration.
• If using for bioassays, ensure the protein is fully dissolved and equilibrated to room temperature before use.
• For cell culture, confirm that the final buffer composition is compatible with your cells and does not contain toxic additives.

This protocol ensures maximum protein stability and activity for cell culture applications.