Recombinant Human IGFBP-6

Recombinant Human IGFBP-6 is used in research applications to investigate its multifaceted roles in cell biology, immune regulation, redox biology, and disease mechanisms, both dependent and independent of IGF-II signaling.

Key scientific applications and rationale include:

• Immune Modulation and Chemotaxis: IGFBP-6 induces chemotaxis of monocytes and T cells, enhances neutrophil oxidative burst and degranulation, and drives metabolic reprogramming in dendritic cells. These properties make it valuable for studying immune cell migration, activation, and inflammatory responses, particularly in autoimmune diseases and cancer.

• Redox Biology and Oxidative Stress: IGFBP-6 influences redox-sensitive processes, including mitochondrial metabolism and the oxidative burst in neutrophils. It is implicated in tissue repair, fibrosis, and immune cell activation, making it relevant for research on oxidative stress-related diseases and redox homeostasis.

• Cell Proliferation, Differentiation, and Apoptosis: IGFBP-6 regulates cell proliferation and apoptosis through both IGF-II-dependent and independent mechanisms. For example, it inhibits proliferation in certain cancer cell lines and modulates differentiation markers in muscle progenitor cells, supporting studies in cancer biology, tissue regeneration, and developmental biology.

• Fibrosis and Tissue Remodeling: IGFBP-6 is involved in the progression of fibrosis in various tissues, including dermal, renal, hepatic, and cardiac systems. Its expression levels and activity can serve as biomarkers or therapeutic targets in fibrotic diseases.

• Cancer Research: IGFBP-6 acts as an inhibitor of IGF-II, suppressing growth and invasion in IGF-II-dependent cancers. Recombinant IGFBP-6 has been shown to decrease proliferation and invasion in nasopharyngeal carcinoma cell lines, highlighting its utility in oncology research.

• Experimental Control and Mechanistic Studies: Recombinant protein allows precise control over concentration and timing in cell culture or animal models, enabling mechanistic studies of IGFBP-6’s effects on signaling pathways, gene expression, and cellular behavior.

Best practices for using recombinant IGFBP-6 include:

• Selecting appropriate concentrations based on published dose-response data (e.g., 0.1–1 μg/mL for immune cell assays; 100 ng/mL for cancer cell proliferation studies).
• Validating activity in your specific cell type or assay system, as effects may be context-dependent.
• Including controls for IGF-II-dependent and independent mechanisms to dissect specific pathways.

In summary, recombinant human IGFBP-6 is a versatile tool for dissecting its roles in immunity, redox biology, cell differentiation, fibrosis, and cancer, enabling targeted mechanistic and translational research across multiple biomedical fields.

Yes, recombinant human IGFBP-6 can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common practice in quantitative ELISA protocols for IGFBP-6, where recombinant proteins serve as standards to generate calibration curves.

Key considerations for using recombinant IGFBP-6 as an ELISA standard:

• Purity and Characterization: The recombinant IGFBP-6 should be highly purified and well-characterized. Impurities or degradation products can affect the accuracy of quantification.
• Concentration Determination: The protein concentration must be precisely measured, typically using absorbance at 280 nm, BCA assay, or HPLC.
• Standard Curve Preparation: Prepare serial dilutions of the recombinant IGFBP-6 in the same buffer as your samples to generate a standard curve covering the expected concentration range in your assay (e.g., 0.062–10 ng/mL).
• Matrix Effects: Ideally, dilute the standard in a matrix similar to your samples (e.g., serum, plasma, or sample diluent) to minimize matrix effects and ensure comparable binding and detection.
• Lot-to-Lot Consistency: Use the same lot of recombinant protein for all assays in a study to maintain consistency. If switching lots, revalidate the standard curve.
• Validation: Confirm that the recombinant standard behaves similarly to endogenous IGFBP-6 in your assay system. Recovery experiments and parallelism tests are recommended.

Scientific best practices:

• Most commercial IGFBP-6 ELISA kits use recombinant human IGFBP-6 as the standard, often expressed in mammalian cells (e.g., NS0 or CHO) to ensure proper folding and post-translational modifications.
• Recombinant standards are suitable for both calibration and quantification, as long as they are compatible with the antibodies used in your ELISA (i.e., recognized by both capture and detection antibodies).
• Always follow the manufacturer’s instructions for reconstitution and dilution of the standard, as protocols may be lot-specific.

Summary Table: Recombinant IGFBP-6 as ELISA Standard

In conclusion: Recombinant human IGFBP-6 is suitable for use as a standard in ELISA quantification and calibration, provided best practices for protein standards are followed.

Research Applications of Recombinant Human IGFBP-6

Recombinant human IGFBP-6 has been validated across multiple research applications, with the most extensively documented uses centered on cancer biology and cellular differentiation studies.

Cancer Research and Tumor Suppression

The most prominent application involves investigating IGFBP-6's tumor-suppressive properties across various cancer models. Recombinant human IGFBP-6 has been used to demonstrate dose-dependent inhibition of cancer cell proliferation, with studies showing maximal inhibitory effects at concentrations around 100 ng/mL over 72-hour exposure periods. This protein has been applied in research examining nasopharyngeal carcinoma, rhabdomyosarcoma, neuroblastoma, lung cancer, colon cancer, prostate cancer, breast cancer, and ovarian cancer. In these applications, recombinant IGFBP-6 has been used to elucidate mechanisms of IGF-II-dependent growth inhibition and to identify signaling pathways such as the GSK3β/β-catenin/cyclin D1 pathway involved in metastasis suppression.

Cellular Differentiation and Stem Cell Studies

Recombinant human IGFBP-6 has been validated for investigating muscle commitment and differentiation of human mesenchymal stem cells. When added to muscle differentiation media, the recombinant protein was taken up by differentiating cells and induced positive feedback effects on intracellular IGFBP-6 detection, demonstrating its utility in studying extracellular signaling during myogenic differentiation.

Bioassay Applications

The protein has been applied in bioassay formats to assess cellular responses, including proliferation assays such as MTS assays for measuring dose-dependent effects on cell growth. These applications have established recombinant IGFBP-6 as a functional tool for quantifying IGF-II-dependent and IGF-independent cellular responses.

Diagnostic and Prognostic Marker Research

Recombinant IGFBP-6 has supported research validating this protein as a potential biomarker for cancer prognosis and diagnosis, with studies identifying it as an independent prognostic indicator for locoregional relapse and distant metastasis in nasopharyngeal carcinoma patients.

To reconstitute and prepare Recombinant Human IGFBP-6 protein for cell culture experiments, dissolve the lyophilized protein in sterile water or buffer to a concentration of at least 100 µg/mL, then dilute to your desired working concentration using cell culture-compatible buffer or medium.

Detailed protocol:

• Reconstitution:

  • Add sterile, high-resistivity water (18 MΩ-cm H₂O) directly to the lyophilized IGFBP-6 protein to achieve a concentration of ≥100 µg/mL.
  • Alternatively, some protocols recommend reconstituting in sterile phosphate-buffered saline (PBS) at 100 µg/mL.
  • Gently pipette up and down or swirl to dissolve. Avoid vigorous vortexing to prevent protein denaturation.

• Dilution:

  • Once fully dissolved, further dilute the stock solution to your desired working concentration using sterile PBS or cell culture medium compatible with your experimental system.
  • If your application is sensitive to carrier proteins, use carrier-free protein and avoid adding BSA or HSA unless stability is a concern.

• Storage:

  • Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
  • Store aliquots at −20 °C or −80 °C, as recommended by the manufacturer, and avoid multiple freeze-thaw cycles to maintain protein integrity.

• General notes:

  • Always use sterile technique to prevent contamination.
  • If the protein was lyophilized from a solution containing volatile acids (e.g., TFA), ensure complete dissolution and, if necessary, buffer exchange before adding to cells.
  • For cell culture, confirm that the final buffer composition is compatible with your cells (e.g., isotonic, pH 7.2–7.4).

Summary Table:

References:

• Reconstitute in sterile water or PBS at ≥100 µg/mL.
• Dilute further in compatible buffer or medium.
• Avoid repeated freeze-thaw cycles.

If your experiment requires a specific buffer (e.g., for bioassays or ELISA), consult the assay protocol for optimal conditions. For most cell culture applications, PBS or serum-free medium is suitable.