Recombinant Human Tie-1

Recombinant Human Tie-1 is used in research applications to study its critical roles in vascular biology, particularly in angiogenesis, endothelial cell function, inflammation, and cancer metastasis. Its use enables precise mechanistic studies, assay development, and therapeutic target validation.

Key reasons to use Recombinant Human Tie-1 in research:

• Modeling Tie-1 Function: Tie-1 is an endothelial-specific receptor tyrosine kinase involved in vascular development, remodeling, and maintenance. Recombinant Tie-1 allows for controlled in vitro and in vivo studies of its signaling pathways and interactions with ligands or co-receptors, such as Tie2.
• Angiogenesis and Vascular Remodeling: Tie-1 modulates angiopoietin/Tie2 signaling, influencing endothelial cell proliferation, migration, and vessel stability. Recombinant Tie-1 can be used to dissect these processes, screen for modulators, or develop functional assays.
• Inflammation and Endothelial Activation: Tie-1 upregulates cell adhesion molecules (VCAM-1, ICAM-1, E-selectin) and proinflammatory gene expression, impacting leukocyte recruitment and vascular inflammation. Recombinant protein enables studies on endothelial activation and inflammatory signaling.
• Cancer and Metastasis Research: Tie-1 is implicated in tumor angiogenesis and metastasis. Blocking Tie-1 function reduces metastatic spread in preclinical models, highlighting its value as a therapeutic target. Recombinant Tie-1 is essential for antibody screening, mechanistic studies, and biomarker development.
• Ligand Identification and Binding Studies: As an orphan receptor, Tie-1’s endogenous ligands are not fully characterized. Recombinant Tie-1 is used in ligand screening, binding assays, and structural studies to identify novel interactions.
• Assay Development: Recombinant Tie-1 is used as a standard or capture protein in ELISA, Western blot, and cell-based assays to quantify Tie-1 or its ligands, or to study downstream signaling events.

Best practices:

• Use carrier-free recombinant Tie-1 for cell-based assays to avoid interference from stabilizers.
• Validate recombinant protein activity and specificity in your assay system.
• Employ appropriate controls, such as Tie-2 or mutant Tie-1, to dissect pathway specificity.

Summary:
Using recombinant human Tie-1 enables detailed investigation of its biological roles, supports drug discovery, and facilitates the development of diagnostic and therapeutic strategies targeting vascular and inflammatory diseases, as well as cancer metastasis.

Yes, you can use recombinant human Tie-1 as a standard for quantification or calibration in your ELISA assays, provided it is properly validated and compatible with your assay system.

Key considerations and supporting details:

• Recombinant proteins are commonly used as ELISA standards: For quantitative ELISA, a standard curve is typically generated using a purified or recombinant form of the target protein, such as recombinant human Tie-1. This allows for precise calculation of Tie-1 concentrations in your samples by comparing their signal to the standard curve.

• Assay compatibility: Most commercial Tie-1 ELISA kits are designed to detect both natural and recombinant human Tie-1, and their standard curves are often generated using recombinant Tie-1 protein. Always confirm that your specific ELISA kit or custom assay is validated to recognize the recombinant form you intend to use.

• Standard preparation: The recombinant Tie-1 should be of high purity and accurately quantified. It is typically reconstituted and serially diluted to generate a standard curve covering the expected concentration range of your samples. Follow the manufacturer's or established protocols for reconstitution and dilution to ensure consistency and accuracy.

• Validation and recovery: If you are using a recombinant Tie-1 standard not supplied with your ELISA kit, it is important to validate its performance. This can be done by spike-and-recovery experiments to ensure that the recombinant standard behaves similarly to endogenous Tie-1 in your sample matrix. Acceptable recovery is generally within ±25% of the expected value.

• Assay-specific requirements: Some ELISA kits are optimized for native Tie-1 and may not detect recombinant forms equally well, depending on epitope recognition. Always check the kit documentation or perform preliminary validation if using a non-kit standard.

• Data analysis: Use appropriate curve-fitting methods (e.g., 4-parameter logistic regression) for standard curve generation and quantification.

Summary of best practices:

• Use a high-purity, well-characterized recombinant Tie-1 protein.
• Validate that your ELISA detects the recombinant standard equivalently to endogenous Tie-1.
• Prepare the standard curve according to established protocols.
• Perform spike-and-recovery experiments if using a custom or third-party recombinant standard.

If these conditions are met, recombinant human Tie-1 is suitable as a standard for ELISA quantification and calibration.

Research Applications of Recombinant Human Tie-1

Recombinant human Tie-1 has been validated for several important research applications in published studies, primarily centered on understanding its role in the angiopoietin-Tie signaling axis and its therapeutic potential.

Vascular and Angiogenic Studies

Recombinant Tie-1 proteins have been utilized to investigate the angiopoietin/Tie receptor system's control over developmental and tumor angiogenesis, inflammatory vascular remodeling, and vessel leakage. Research has demonstrated that Tie-1 functions as a modulator of Tie-2 receptor activity, making it valuable for studying how these receptors coordinate vascular responses. The protein has been employed in experiments examining endothelial cell proliferation and survival pathways, with studies using recombinant Tie-1 to elucidate signal transduction mechanisms involving PI 3-kinase binding and Akt phosphorylation.

Lymphatic Endothelial Cell Migration

Recent research has validated recombinant Tie-1 for studying lymphatic endothelial cell (LEC) biology. Specifically, investigations have demonstrated that Tie-1 binds to ligands such as Polydom/SVEP1, which facilitates migration of lymphatic endothelial cells. This application has expanded understanding of how the Tie-1 receptor regulates lymphatic vascular function.

Therapeutic Target Validation

Recombinant Tie-1 has been instrumental in validating Tie-1 as a therapeutic target. Research has shown that Tie-1 deletion or function-blocking approaches can inhibit tumor angiogenesis, delay tumor growth, prevent metastatic cell extravasation, and reduce metastatic foci formation. These studies have established the scientific rationale for developing Tie-1-targeted therapeutics for cancer applications.

Biochemical and Structural Studies

The protein has been used in biochemical assays to demonstrate direct binding interactions, such as association with p85/PI 3-kinase in vitro, and to characterize autophosphorylation-dependent signaling mechanisms. These applications have been critical for understanding the molecular basis of Tie-1 function in endothelial cell biology.

To reconstitute and prepare Recombinant Human Tie-1 protein for cell culture experiments, dissolve the lyophilized protein in sterile buffer (commonly sterile PBS or water) at a concentration of at least 100 μg/mL, then gently mix and further dilute as needed for your assay.

Step-by-step protocol:

• Centrifuge the vial briefly (3000–3500 rpm, 5 min) before opening to collect all powder at the bottom.
• Add sterile buffer: Use sterile PBS or water, as recommended by the product datasheet or certificate of analysis. For Tie-1, reconstitution at 100 μg/mL is standard. For example, add 100 μL buffer to a 100 μg vial.
• Gentle mixing: Do not vortex. Gently swirl or pipette up and down to dissolve. Let stand at room temperature for 15–30 min to ensure complete solubilization.
• Dilution: After initial reconstitution, dilute the protein to your working concentration using cell culture medium or PBS. For stability, include a carrier protein (e.g., 0.1%–1% BSA, FBS, or HSA) in the dilution buffer unless your experiment requires serum-free conditions.
• Aliquoting and storage: Prepare aliquots sized for single use (≥20 μL) to avoid repeated freeze-thaw cycles, which reduce activity. Store aliquots at −20 °C to −80 °C for long-term storage. For short-term use (≤1 week), store at 2–8 °C.
• Special considerations: If performing serum-free culture or in vivo work, avoid animal-derived carrier proteins; use trehalose as a stabilizer if needed.

Key technical notes:

• Avoid vigorous agitation (no vortexing) to prevent protein denaturation.
• Check the product datasheet for buffer composition (e.g., MES, Tris, NaCl, sucrose) and adjust your reconstitution buffer accordingly if specific requirements are listed.
• Typical working concentrations for cell culture assays range from 0.1–1.0 mg/mL for stock solutions, with further dilution as required for your experimental design.

Summary Table:

Always consult the specific product datasheet for any unique requirements. This protocol ensures optimal solubility and biological activity of recombinant Tie-1 protein for cell culture applications.