Recombinant Human Tie-2

Recombinant Human Tie-2 is widely used in research applications to study and manipulate the Angiopoietin-Tie signaling axis, which is essential for vascular biology, angiogenesis, and endothelial cell function. Using recombinant Tie-2 enables precise investigation of ligand-receptor interactions, downstream signaling, and therapeutic modulation in both basic and translational research.

Key reasons to use Recombinant Human Tie-2 in research:

• Ligand Binding Studies: Recombinant Tie-2, often as a soluble Fc or His-tagged chimera, is used to characterize binding affinities and specificities of natural ligands (such as Angiopoietin-1 and Angiopoietin-2) or engineered variants. This is critical for dissecting the molecular mechanisms of Tie-2 activation and inhibition.

• Therapeutic Target Validation: Tie-2 is a validated target for diseases involving vascular dysfunction, including sepsis, cancer, diabetic retinopathy, and chronic inflammation. Recombinant Tie-2 allows for screening and characterization of agonists, antagonists, or antibodies that modulate its activity, supporting drug discovery and preclinical development.

• Functional Assays: Recombinant Tie-2 can be used in cell-based assays to assess downstream effects such as receptor phosphorylation, endothelial cell migration, tube formation, and barrier integrity. These assays help elucidate the biological consequences of Tie-2 activation or inhibition.

• ELISA and Quantification: Recombinant Tie-2 serves as a standard or capture reagent in ELISA and other immunoassays to quantify ligand levels or monitor receptor-ligand interactions in biological samples.

• Decoy Receptor Applications: Soluble recombinant Tie-2 can function as a decoy receptor, sequestering angiopoietins and modulating signaling in vitro or in vivo, which is useful for mechanistic studies and therapeutic intervention models.

• Species Cross-Reactivity and Conservation: The high sequence conservation of Tie-2 between human and murine species allows recombinant human Tie-2 to be used in both human and mouse model systems, facilitating translational research.

• Vascular Biology and Disease Modeling: Tie-2 signaling is central to vascular stability, endothelial barrier function, and anti-inflammatory responses. Recombinant Tie-2 is instrumental in modeling these processes and testing interventions in disease-relevant contexts.

In summary, recombinant human Tie-2 is a versatile tool for dissecting vascular signaling pathways, validating therapeutic targets, and developing new interventions for vascular-related diseases.

Yes, recombinant Human Tie-2 protein can be used as a standard for quantification or calibration in ELISA assays, provided it is properly purified and quantified.

Essential context and supporting details:

• Standard Curve Requirement: Quantitative ELISA assays require a standard curve generated from known concentrations of the target protein. Recombinant proteins are commonly used for this purpose, especially when purified native protein is unavailable.
• Parallel Recognition: ELISA kits designed for Tie-2 typically use antibody pairs that recognize both recombinant and natural forms of Human Tie-2 in a parallel manner, ensuring that the standard curve generated from recombinant protein is valid for quantifying endogenous Tie-2 in biological samples.
• Kit Protocols: Commercial Human Tie-2 ELISA kits routinely include recombinant Human Tie-2 as the calibration standard, confirming its suitability for quantification.
• Preparation Guidelines: For best results, the recombinant protein should be highly purified, accurately quantified (e.g., by HPLC or spectrophotometry), and prepared fresh or stored appropriately to maintain stability.
• Validation: It is important to validate that the recombinant standard behaves similarly to the endogenous protein in your specific assay system. This can be confirmed by recovery experiments and parallelism testing.

Additional relevant information:

• Assay Sensitivity and Range: Ensure the concentration range of your recombinant standard matches the dynamic range and sensitivity of your ELISA assay.
• Source and Isoform: Confirm that the recombinant Tie-2 standard matches the isoform and post-translational modifications (if relevant) of the Tie-2 you expect in your samples, as differences can affect antibody recognition and quantification accuracy.
• Documentation: Record the lot number, concentration, and preparation method of your recombinant standard for reproducibility and troubleshooting.

In summary, recombinant Human Tie-2 is scientifically appropriate as an ELISA standard, provided it is well-characterized and validated for your assay conditions.

Recombinant Human Tie-2 has been validated for several key applications in published research, primarily as a molecular tool for studying angiopoietin-Tie2 signaling, as a therapeutic target, and as a reagent in biochemical and cell-based assays.

Validated Applications in Published Research:

• Ligand Binding and Receptor Activation Assays:
  Recombinant Tie-2 is widely used to assess the binding affinity and specificity of angiopoietin ligands (such as Ang1 and Ang2) and engineered variants. For example, soluble recombinant Tie2 (sTie2) has been used in pull-down assays, ELISA, and surface plasmon resonance to quantify ligand-receptor interactions and to screen for enhanced binding variants.

• Functional Cell-Based Assays:
  Recombinant Tie-2 is employed in cellular assays to study downstream signaling events, such as Tie2 phosphorylation, endothelial cell migration, tube formation, and barrier function. These assays help elucidate the biological effects of Tie2 activation or inhibition in endothelial cells.

• Therapeutic Target Validation:
  Recombinant Tie-2 has been used in preclinical models to validate its role as a therapeutic target in diseases characterized by vascular dysfunction, including sepsis, cancer, ischemic diseases, and chronic inflammatory disorders. Studies have shown that activating Tie2 with recombinant ligands or agonistic antibodies can reduce vascular leak, inflammation, and organ damage in animal models.

• ELISA and Western Blot (WB):
  The extracellular domain of recombinant human Tie-2 is used as a standard or capture reagent in ELISA and as a positive control in Western blotting to detect Tie2 or its ligands in biological samples.

• Protein Engineering and Structural Studies:
  Recombinant Tie-2 is utilized in structural biology to resolve the molecular interface with its ligands, enabling rational design of high-affinity variants and therapeutic antibodies.

Summary Table: Applications of Recombinant Human Tie-2

Additional Notes:

• Recombinant Tie-2 is often used as a soluble Fc-fusion protein (Tie2-Fc) to facilitate purification and detection.
• It is also used as a molecular decoy to sequester angiopoietins in functional inhibition studies.
• While most studies focus on endothelial biology, Tie2 has also been investigated in tumor biology and other tissue contexts.

If you require protocols or more specific assay details, please specify the application of interest.

To reconstitute and prepare Recombinant Human Tie-2 protein for cell culture experiments, dissolve the lyophilized protein in sterile water or buffer as recommended in the product datasheet, typically using sterile water for initial reconstitution. Gently mix by swirling or inverting; avoid vigorous shaking or vortexing to prevent protein denaturation and foaming.

Step-by-step protocol:

• Check formulation: Most recombinant Tie-2 proteins are supplied lyophilized, often with stabilizers such as trehalose and mannitol in PBS. Confirm the formulation in your product datasheet.
• Reconstitution: Add sterile water (or the recommended buffer) to the vial. The volume depends on the desired final concentration; for example, if the vial contains 10 μg, and you want a 0.1 mg/mL stock, add 100 μL sterile water.
• Mixing: Allow the vial to sit at room temperature for 15–30 minutes with gentle agitation (e.g., slow inversion or gentle pipetting). Do not vortex.
• Aliquoting: Once fully dissolved, aliquot the solution into small volumes to avoid repeated freeze-thaw cycles, which can degrade the protein.
• Storage: Store aliquots at −20°C or below for long-term storage. For short-term use (2–7 days), keep at 4–8°C.
• Working solution: Before adding to cell culture, dilute the stock solution into your cell culture medium to the desired working concentration. Ensure the final buffer conditions are compatible with your cells (e.g., isotonicity, pH 7.2–7.4).

Additional considerations:

• If the protein is difficult to dissolve, allow more time at 4°C or room temperature, and gently pipette up and down.
• Avoid repeated freeze-thaw cycles by preparing single-use aliquots.
• Confirm the endotoxin level is suitable for cell culture (<0.1 EU/μg is typical for sensitive applications).
• If the protein contains tags (e.g., His, Fc, GST), ensure these do not interfere with your experimental design.

Example calculation:If your vial contains 10 μg Tie-2 and you want a 100 μg/mL stock:

• Add 100 μL sterile water.
• Mix gently and let dissolve.
• Aliquot into 10–20 μL portions and freeze at −20°C.

Summary of best practices:

• Use sterile technique throughout.
• Avoid vigorous mixing.
• Aliquot and freeze to minimize degradation.
• Dilute into cell culture medium immediately before use.

This protocol ensures optimal protein stability and activity for cell culture experiments. Always consult the specific product datasheet for any unique instructions.