Recombinant Mouse Tie-2

Recombinant mouse Tie-2 protein serves as a valuable research tool for investigating the angiopoietin-Tie signaling axis, which is fundamental to vascular biology and endothelial cell function. Here are the key reasons to incorporate this protein into your research:

Vascular Biology Applications

Recombinant mouse Tie-2 enables direct study of the angiopoietin-Tie signaling system, a vascular-specific receptor tyrosine kinase pathway essential for normal vascular development. This protein is particularly useful for examining receptor-ligand interactions, as it allows you to assess binding kinetics and signaling activation in controlled experimental conditions. The recombinant form provides a standardized, reproducible substrate for investigating how angiopoietin ligands (particularly Ang1 and Ang2) interact with and activate the Tie2 receptor.

Mechanistic Research

Using recombinant mouse Tie-2 facilitates investigation of the dual functions of Tie2 signaling in both vascular growth and vascular maintenance. This is particularly important for understanding how Tie2 activation promotes endothelial barrier function, vascular stability, and endothelial cell survival. The protein can be employed in binding assays, phosphorylation studies, and cell-based assays to elucidate downstream signaling pathways activated by Tie2 engagement.

Disease Model Development

The recombinant protein is instrumental for developing and validating therapeutic approaches targeting vascular dysfunction. Since Tie2 receptors are expressed primarily on endothelial and hematopoietic progenitor cells and play critical roles in angiogenesis and vasculogenesis, this tool enables you to model disease states involving vascular leakage, inflammatory responses, and endothelial barrier dysfunction. This makes it particularly relevant for research into sepsis, influenza, diabetic complications, and other conditions characterized by vascular instability.

Protein Engineering and Optimization

Recombinant mouse Tie-2 serves as a benchmark for evaluating engineered Tie2 agonists and variants. By using this standardized protein, you can quantitatively measure improvements in binding affinity and receptor activation efficiency of novel therapeutic candidates, supporting structure-activity relationship studies and rational drug design efforts.

You can use recombinant mouse Tie-2 as a standard for quantification or calibration in ELISA assays, provided it is highly purified and its concentration is accurately known. This is a common and accepted practice in quantitative ELISA development and commercial kits.

Key considerations and supporting details:

• Commercial ELISA kits for mouse Tie-2 routinely use recombinant mouse Tie-2 as the standard for generating the calibration curve. These kits demonstrate that recombinant Tie-2 produces standard curves that are parallel to those generated with natural Tie-2, supporting its suitability for quantification.
• Assay specificity: The ELISA must be validated to ensure that the recombinant Tie-2 standard is recognized equivalently to the endogenous (natural) protein in your samples. Most commercial kits confirm this by showing parallelism between standard curves generated with recombinant and natural proteins.
• Protein quality: The recombinant Tie-2 should be highly purified, and its concentration must be accurately determined (e.g., by absorbance at 280 nm or amino acid analysis). Impurities or inaccurate quantification can lead to errors in your standard curve.
• Standard preparation: Prepare a serial dilution of the recombinant Tie-2 in the same buffer or matrix as your samples to minimize matrix effects. Follow best practices for pipetting and mixing to ensure reproducibility.
• Carrier proteins: If your recombinant Tie-2 is supplied carrier-free, it is suitable for use as a standard. If it contains carrier proteins (e.g., BSA), ensure these do not interfere with your assay.
• Documentation: Record the source, lot, and concentration of the recombinant protein used for traceability and reproducibility.

Limitations:

• The recombinant standard should match the form of Tie-2 detected by your assay (e.g., full-length, extracellular domain, or tagged variants).
• For regulatory or diagnostic applications, additional validation may be required. For research use, this approach is standard practice.

Summary Table:

In conclusion:
Recombinant mouse Tie-2 is widely used as a standard for ELISA quantification, provided it is pure, accurately quantified, and validated for your assay format. Always verify that your specific ELISA detects the recombinant form equivalently to the endogenous protein.

Recombinant Mouse Tie-2 has been validated in published research primarily for applications in biochemical binding assays, cell signaling studies, and in vivo disease models focused on vascular biology and inflammation.

Key validated applications include:

• Protein–protein interaction assays: Recombinant Mouse Tie-2 (often as a soluble extracellular domain, sTie2) is used to assess binding affinity and complex formation with ligands such as Angiopoietin-1 (Ang1) and its engineered variants. These assays typically involve size exclusion chromatography and SDS-PAGE to confirm stable complex formation between Tie2 and its ligands.

• Cell signaling studies: Recombinant Tie-2 is used in cellular assays to evaluate downstream signaling events, such as Tie2 phosphorylation, which is critical for endothelial cell function, migration, and tube formation. These studies help elucidate the role of Tie2 activation in maintaining endothelial barrier integrity and preventing vascular leakage.

• In vivo disease models: Recombinant Tie-2 and its ligands have been applied in mouse models of sepsis and vascular dysfunction to investigate therapeutic potential. For example, enhanced Tie2 activation by engineered Ang1 variants was shown to reduce inflammatory cytokines and attenuate organ damage in septic mice, demonstrating the utility of recombinant Tie-2 in preclinical therapeutic validation.

• ELISA and standardization: Recombinant Mouse Tie-2 is commonly used as a standard or capture reagent in ELISA assays to quantify ligand binding or to measure Tie2 levels in biological samples.

• Vascular biology research: Studies utilize recombinant Tie-2 to dissect its role in angiogenesis, vessel maturation, and endothelial cell survival, both in vitro and in vivo, including genetic and pharmacological manipulation of the Tie2 pathway.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies, highlighting the versatility of recombinant Mouse Tie-2 in vascular and inflammation research. If you require protocols or more technical details for a specific application, please specify the intended use.

To reconstitute and prepare Recombinant Mouse Tie-2 protein for cell culture experiments, dissolve the lyophilized protein in sterile water or buffer, then dilute with a carrier protein solution such as 0.1% BSA or 10% FBS to stabilize and prevent adsorption. Avoid vigorous agitation and aliquot for storage at −20°C to −80°C.

Step-by-step protocol:

• Reconstitution:

  • Allow the vial to equilibrate to room temperature before opening to prevent condensation.
  • Add sterile, distilled water or the recommended buffer (check the product datasheet for specific requirements; PBS or HEPES-buffered saline are common).
  • Gently mix by inverting or tapping; avoid vortexing or vigorous shaking to prevent protein denaturation and foaming.
  • Let the solution sit for 15–30 minutes at room temperature with gentle agitation until fully dissolved.
  • If particulates remain, allow the solution to stand at 4°C for up to 2 hours or gently pipette to aid dissolution.

• Stabilization and Dilution:

  • Dilute the reconstituted protein with a carrier protein-containing solution (e.g., 0.1% BSA, 10% FBS, or 5% HSA) to minimize adsorption and loss.
  • For working concentrations, prepare stock solutions (e.g., 50–100 μg/mL) in the carrier protein solution.
  • Filter sterilize if necessary using a 0.2 μm filter.

• Aliquot and Storage:

  • Aliquot the solution to avoid repeated freeze-thaw cycles.
  • Store aliquots at −20°C to −80°C for long-term stability.
  • Optionally, add 5–50% glycerol for enhanced stability during storage.

• Preparation for Cell Culture:

  • Thaw aliquots on ice and use immediately; avoid repeated freeze-thaw cycles.
  • Dilute to the desired final concentration in cell culture medium immediately before use.

Additional notes:

• Always consult the specific product datasheet for recommended reconstitution buffer, concentration, and storage conditions, as these may vary depending on protein formulation and intended application.
• For functional assays, confirm protein activity after reconstitution using appropriate controls.

This protocol ensures optimal solubility, stability, and biological activity of recombinant Mouse Tie-2 protein for cell culture experiments.