Recombinant Mouse IGFBP-6

Recombinant mouse IGFBP-6 offers several compelling advantages for research applications, particularly in studies involving insulin-like growth factor (IGF) signaling, cell differentiation, and cancer biology.

IGF-II Regulation and Cancer Research

Recombinant mouse IGFBP-6 is uniquely valuable because it demonstrates marked binding preference for IGF-II over IGF-I. This specificity makes it an excellent tool for investigating IGF-II-dependent biological processes. The protein acts as a potent inhibitor of IGF-II receptor interactions, effectively blocking major IGF-II functions including proliferation, differentiation, cell adhesion, and colony formation.

In cancer research applications, IGFBP-6 has demonstrated significant inhibitory effects across multiple tumor types. It decreases cancer cell proliferation and survival by inhibiting IGF-II effects, and has shown particular efficacy in suppressing growth of IGF-II-dependent cancers including neuroblastoma, rhabdomyosarcoma, colon cancer, lung cancer, prostate cancer, breast cancer, and ovarian cancer. Studies have shown that exogenous recombinant IGFBP-6 dose-dependently inhibits cell proliferation in vitro and dramatically suppresses xenograft growth in vivo.

Skeletal Muscle and Stem Cell Differentiation

Recombinant mouse IGFBP-6 is particularly useful for studying muscle stem cell biology. When added to culture media, it increases both pluripotency-associated markers (OCT4, SOX2) and muscle differentiation markers (MyoD, MyoG, MHC) at early time points. The protein promotes progenitor cell commitment to the muscle lineage while simultaneously maintaining a primitive progenitor phenotype, making it valuable for investigating the temporal dynamics of myogenic differentiation.

IGF-Independent Functions

Beyond IGF regulation, recombinant mouse IGFBP-6 exhibits important IGF-independent effects, including inhibition of angiogenesis and modulation of cell migration. These pleiotropic functions expand its utility for studying diverse biological processes beyond the classical IGF signaling axis.

Practical Research Advantages

The protein is widely available in both carrier-free and carrier-protein formulations, allowing researchers to select the appropriate preparation for their specific experimental needs, whether for cell culture supplementation, ELISA standards, or Western blot applications. Its expression across multiple tissues including lung, liver, and gut makes it relevant for investigating tissue-specific IGF signaling in various physiological contexts.

Yes, recombinant mouse IGFBP-6 can be used as a standard for quantification and calibration in ELISA assays, provided it is appropriately characterized and validated for your specific application.

Standard Preparation and Use

Recombinant proteins expressed in mammalian systems (such as NSO cells) are commonly employed as calibration standards in sandwich ELISA formats. When using recombinant mouse IGFBP-6 as your standard, the protein should be:

Properly characterized - The recombinant standard should have a defined amino acid sequence corresponding to the target protein region (typically the mature, biologically active form). Documentation of the expression system, purification method, and protein identity is essential for assay validation.

Concentration verified - Accurately determine the concentration of your recombinant standard using orthogonal methods such as absorbance spectrophotometry (A280), amino acid analysis, or quantitative amino acid composition analysis before preparing your standard curve.

Standard Curve Development

To establish a valid calibration curve, prepare serial dilutions of your recombinant mouse IGFBP-6 standard across the expected measurement range. The standard curve must be generated fresh for each assay run to ensure quantitative accuracy. A well-constructed standard curve should demonstrate linearity across your working range, typically with correlation coefficients (r²) ≥ 0.98.

Critical Validation Considerations

Matrix effects - Test your recombinant standard in the same diluent buffer and sample matrix (serum, plasma, or cell culture supernatant) that you will use for experimental samples. Internal controls prepared by spiking known amounts of recombinant IGFBP-6 into your actual sample matrix help confirm that the standard curve accurately predicts analyte recovery in your specific biological samples.

Specificity confirmation - Verify that your detection antibodies (capture and detection) specifically recognize mouse IGFBP-6 without significant cross-reactivity to other IGFBP family members or related proteins.

Assay precision - Replicate measurements of your standard at multiple concentrations (typically in duplicate or triplicate) to establish intra-assay and inter-assay coefficient of variation (CV). Acceptable precision is generally <10% intra-assay CV and <12% inter-assay CV.

Practical Recommendations

Prepare your recombinant standard in the appropriate diluent buffer provided with your ELISA kit or a validated alternative (such as PBS or TBS). Store concentrated stock solutions appropriately to maintain protein stability and activity. Avoid repeated freeze-thaw cycles, which can compromise protein integrity and lead to inaccurate quantification.

Recombinant Mouse IGFBP-6 has been validated for several applications in published research, primarily in the context of functional studies, biomarker analysis, and mechanistic investigations in mouse models and cell-based assays.

Validated Applications in Published Research:

• In vivo functional studies: Recombinant mouse IGFBP-6 (rIGFBP6) has been administered to mice to study its effects on immune regulation, particularly in sepsis models. For example, rIGFBP6 was injected intraperitoneally in cecal ligation and puncture (CLP) models to assess its impact on survival, macrophage trafficking, and cytokine expression. These studies demonstrated that rIGFBP6 acts as a negative regulator of macrophage migration by suppressing epithelial CCL2 secretion, with downstream effects on bacterial clearance and organ injury.

• ELISA (Enzyme-Linked Immunosorbent Assay): Recombinant mouse IGFBP-6 is widely used as a standard or positive control in ELISA kits designed to quantify IGFBP-6 in mouse serum, plasma, or cell culture supernatants. These assays are used in studies of cancer, fibrosis, and metabolic regulation.

• Western Blot (WB): rIGFBP-6 is validated as a positive control and for detection in Western blotting, enabling the analysis of IGFBP-6 expression in tissues and cell lysates.

• Immunogen: Recombinant IGFBP-6 is used as an immunogen for antibody production and validation.

• SDS-PAGE: The protein is routinely analyzed by SDS-PAGE for purity assessment and molecular weight confirmation.

• Cell-based functional assays: rIGFBP-6 has been used in vitro to study its effects on cell migration, proliferation, and survival, particularly in cancer cell lines and primary cells. It is known to inhibit IGF-II-dependent cancer cell growth (e.g., neuroblastoma, colon cancer, rhabdomyosarcoma) and modulate cell migration in an IGF-independent manner.

• Tissue and disease models: Recombinant mouse IGFBP-6 has been implicated in studies of hepatic fibrosis, where it was identified as a driver of fibrogenesis and a target for therapeutic intervention. It is also studied in the context of redox biology, tissue repair, and immune regulation.

Summary Table of Validated Applications

Key Research Areas:

• Immunomodulation and sepsis: rIGFBP-6 modulates macrophage trafficking and immune responses in murine sepsis models.
• Cancer biology: Inhibits IGF-II-dependent tumor cell proliferation and migration.
• Fibrosis: Identified as a driver of hepatic fibrosis and a target for anti-fibrotic therapies.
• Redox biology and tissue repair: Implicated in oxidative stress responses and tissue remodeling.

These applications are supported by both direct experimental use of recombinant mouse IGFBP-6 and by its role as a standard or control in immunoassays and protein analyses.

To reconstitute and prepare Recombinant Mouse IGFBP-6 protein for cell culture experiments, follow these general guidelines based on manufacturer protocols and best practices:

Reconstitution

1. Reconstitution Buffer:

   • Reconstitute the lyophilized protein in sterile distilled water or sterile PBS (pH 7.4).
   • Typical recommended concentration: 0.1–0.5 mg/mL.
   • Avoid using buffers containing reducing agents (e.g., DTT, β-mercaptoethanol) unless specifically required, as IGFBP-6 is a cysteine-rich protein and may be sensitive to reduction.

2. Procedure:

   • Gently add the appropriate volume of sterile water or PBS to the vial.
   • Allow the vial to sit for a few minutes to let the powder hydrate.
   • Gently swirl or rotate the vial to dissolve the protein; do not vortex, as this may denature the protein.
   • If necessary, let the solution sit at room temperature for 10–15 minutes with occasional gentle mixing.

Preparation for Cell Culture

1. Sterility:

   • Ensure all steps are performed under sterile conditions to avoid contamination.
   • Use sterile pipette tips and vials.

2. Storage:

   • For short-term use (within a week), store the reconstituted protein at 4–8°C.
   • For long-term storage, aliquot the protein and store at –20°C or –80°C. Avoid repeated freeze-thaw cycles.

3. Dilution:

   • Dilute the reconstituted protein in cell culture medium immediately before use.
   • Filter-sterilize the diluted protein solution using a 0.22 µm filter if not already sterile.

4. Concentration:

   • Use the protein at the desired concentration for your experiment (typically in the range of 10–100 ng/mL, but optimize based on your specific assay).

Additional Notes

• Endotoxin: Ensure the protein has low endotoxin levels (<1.0 EU/µg) to avoid unwanted immune responses in cell culture.
• Activity: If the protein’s activity is critical, validate its functionality in a relevant assay (e.g., IGF2 binding or inhibition of cell proliferation).

Example Protocol

1. Reconstitute 10 µg of lyophilized Recombinant Mouse IGFBP-6 in 100 µL sterile PBS (0.1 mg/mL).
2. Gently mix until fully dissolved.
3. For cell culture, dilute to the desired concentration in pre-warmed cell culture medium.
4. Filter-sterilize the final solution.
5. Add to cells and incubate as per your experimental design.

Always refer to the specific product manual for any unique requirements or recommendations.