Recombinant Human IGFBP-5

Recombinant Human IGFBP-5 is used in research applications to investigate its roles in cell survival, fibrosis, tissue regeneration, extracellular matrix (ECM) production, and as a modulator of insulin-like growth factor (IGF) signaling, both in IGF-dependent and IGF-independent contexts.

Key scientific reasons to use recombinant human IGFBP-5 include:

• Cell Survival and Anti-Apoptotic Effects: IGFBP-5 enhances the survival of various cell types, such as hepatic stellate cells and myofibroblasts, by suppressing apoptosis through mechanisms that are often independent of IGF signaling. This makes it valuable for studying cell viability and stress responses in vitro.

• Fibrosis and ECM Regulation: IGFBP-5 promotes the expression of pro-fibrotic genes and ECM components (e.g., collagen Iα1, fibronectin, TIMP1, MMP1), making it a critical factor in models of tissue fibrosis, including liver and lung fibrosis. Recombinant IGFBP-5 can be used to induce or modulate fibrotic responses in cell and tissue culture systems.

• Tissue Repair and Regeneration: IGFBP-5 is implicated in tissue repair processes, such as bone formation and periodontal tissue regeneration, by enhancing cell proliferation, migration, and differentiation of mesenchymal stem cells. It is commonly used in assays investigating osteogenesis, muscle differentiation, and wound healing.

• IGF-Dependent and IGF-Independent Signaling: While IGFBP-5 binds IGF-I and IGF-II, modulating their bioavailability and receptor interactions, it also exerts direct effects on cells independent of IGF signaling, such as influencing cell adhesion, migration, and survival pathways.

• Disease Modeling: Recombinant IGFBP-5 is used to model pathological conditions such as diabetic kidney disease, idiopathic pulmonary fibrosis, and cancer (e.g., glioblastoma), where it is involved in disease progression, ECM remodeling, and cell invasion.

• Experimental Control: Using recombinant protein allows for precise control of IGFBP-5 concentration and timing in experimental systems, facilitating mechanistic studies and the dissection of its specific biological effects.

Typical applications include:

• Cell culture assays for survival, proliferation, and differentiation.
• Induction or inhibition of fibrosis in tissue models.
• Studies of ECM production and remodeling.
• Investigation of IGF signaling modulation.
• Disease modeling in vitro and ex vivo.

Summary: Recombinant human IGFBP-5 is a versatile tool for dissecting its multifaceted roles in cell biology, tissue remodeling, and disease, enabling controlled, reproducible experiments to elucidate its mechanisms of action and therapeutic potential.

Yes, Recombinant Human IGFBP-5 can be used as a standard for quantification or calibration in ELISA assays, provided that the recombinant protein is suitable for your specific assay format and detection system.

Key Points to Consider:

• Purpose of Recombinant Standards: Recombinant proteins like Human IGFBP-5 are commonly used to generate standard curves in ELISA assays. These curves allow you to convert optical density readings into analyte concentrations in your samples.

• Carrier Protein: If the recombinant IGFBP-5 is carrier-free (without BSA or other stabilizing proteins), it is generally preferred for use as an ELISA standard because carrier proteins can sometimes interfere with assay performance or quantification.

• Purity and Activity: Ensure the recombinant protein is of high purity (>95%) and has been validated for immunoreactivity in ELISA. Most commercial recombinant IGFBP-5 proteins (e.g., from R&D Systems, BioLegend, Bio-Techne) are tested for activity and suitability as ELISA standards.

• Matching Assay Type: The recombinant standard should match the form of IGFBP-5 detected by your ELISA (e.g., glycosylated vs. non-glycosylated). Some kits are specifically designed to detect glycosylated forms, so confirm compatibility.

• Diluent and Buffer: Always use the recommended diluent (often provided with the ELISA kit) to prepare your standard curve. Using an inappropriate buffer can affect protein stability and assay accuracy.

Example from Literature and Vendors:

• R&D Systems and BioLegend both offer carrier-free recombinant Human IGFBP-5 and explicitly state that these proteins are suitable for use as ELISA standards.
• Sigma-Aldrich, Fisher Scientific, and other ELISA kit manufacturers typically include a recombinant IGFBP-5 standard in their kits for calibration.

Best Practices:

• Avoid repeated freeze-thaw cycles of the recombinant protein.
• Validate linearity and parallelism of your standard curve with your samples.
• Check for cross-reactivity if your assay detects modified forms of IGFBP-5.

In summary, yes, recombinant Human IGFBP-5 is appropriate for use as a standard in ELISA assays, especially when it is carrier-free and matched to your assay requirements.

Recombinant Human IGFBP-5 has been validated for a diverse range of applications in published research, primarily in the following areas:

• Bioassays: Used to assess biological activity, such as modulation of cell proliferation, differentiation, migration, and senescence in various cell types including mesenchymal stem cells, fibroblasts, keratinocytes, and cancer cells.
• Western Blotting: Employed for protein detection and quantification in cell lysates and tissue extracts.
• ELISA (Enzyme-Linked Immunosorbent Assay): Utilized for quantifying IGFBP-5 protein levels in biological samples, including validation of differential expression in cancer research.
• Radioimmunoassay (RIA): Used for sensitive quantification and validation of IGFBP-5 in biological fluids and tissues.
• Mass Cytometry (CyTOF): Applied for high-dimensional single-cell analysis in spatial biology and cell profiling studies.
• Standard or Calibrator in Quantitative Assays: Used as a reference standard in various quantitative immunoassays.

Key validated research applications include:

• Fibrosis Studies: IGFBP-5 promotes extracellular matrix production and fibrosis in human lung and liver tissues, validated by ex vivo and in vivo bioassays.
• Bone and Osteogenesis Research: Demonstrated to stimulate bone formation and osteoblast differentiation, both IGF-dependent and IGF-independent, using bioassays and cell proliferation assays.
• Cancer Biology: Validated for investigating tumor cell invasion, migration, and metastasis, especially in glioblastoma and breast cancer models, using bioassays, ELISA, and gene editing approaches.
• Regenerative Medicine: Shown to enhance periodontal tissue regeneration and stem cell function in inflammatory niches through bioassays and cell migration/proliferation studies.
• Cell Signaling and Differentiation: Used to study IGF signaling modulation, keratinocyte differentiation, and muscle/kidney development in various cell types.

Summary Table of Validated Applications

Additional validated uses include studies on apoptosis, angiogenesis, wound healing, and tissue regeneration, as well as modulation of IGF activity in various biological systems.

These applications are supported by peer-reviewed publications and are widely used in cell biology, cancer research, regenerative medicine, and tissue engineering.

To reconstitute and prepare Recombinant Human IGFBP-5 protein for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS), pH 7.4, to a concentration of 100 μg/mL (0.1 mg/mL) or within the range of 0.1–1.0 mg/mL as required for your application.

Detailed protocol and best practices:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Add sterile PBS (pH 7.4) directly to the vial to achieve your desired concentration (commonly 0.1–1.0 mg/mL).
• Do not vortex the solution; gently swirl or invert to dissolve the protein completely.
• If your application is sensitive to low protein concentrations or adsorption, consider adding a carrier protein (such as 0.1–1% BSA or HSA) to improve stability, unless the presence of carrier protein would interfere with your experiment.
• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can reduce activity.
• Storage after reconstitution:
  • Short-term (up to 1 week): 2–8 °C
  • Long-term (up to 3 months): –20 °C or lower
  • For extended storage, aliquot and store at –70 °C or lower.
• Avoid repeated freeze/thaw cycles to maintain protein integrity and activity.

Additional notes:

• If the protein was supplied in an acidic buffer (e.g., sodium citrate, pH 4), you may dilute or exchange into PBS, pH 7.4, for cell culture compatibility.
• For sensitive cell types or assays, confirm that the final buffer composition is compatible with your experimental system.
• Always refer to the specific product datasheet for any unique instructions related to your batch or formulation.

Summary Table: IGFBP-5 Reconstitution for Cell Culture

This protocol ensures optimal solubility, stability, and bioactivity of IGFBP-5 for cell culture experiments.