Recombinant Human VEGF R2

Recombinant Human VEGF R2 (also known as VEGFR2 or KDR) is widely used in research applications to study and manipulate angiogenesis, lymphangiogenesis, and related signaling pathways. Its use is justified by several key scientific advantages:

• High-affinity VEGF binding and antagonism: Recombinant soluble VEGFR2 binds VEGF ligands (such as VEGF-A and VEGF-C) with high affinity, acting as a potent antagonist that sequesters VEGF and prevents it from activating cell-surface VEGFR2 receptors. This property is critical for dissecting VEGF-dependent signaling in vitro and in vivo.

• Angiogenesis and lymphangiogenesis research: VEGFR2 is the principal mediator of VEGF-driven angiogenesis, which is essential for blood vessel formation in physiological processes (e.g., wound healing) and pathological conditions (e.g., cancer, diabetic retinopathy, rheumatoid arthritis). Recombinant VEGFR2 enables precise modulation of these pathways, allowing researchers to study vessel growth, remodeling, and inhibition.

• Cancer and metastasis models: Inhibition of VEGFR2 signaling suppresses tumor angiogenesis and growth, making recombinant VEGFR2 a valuable tool for modeling anti-angiogenic therapies and understanding mechanisms of tumor vascularization and metastasis. It is also used to evaluate the efficacy of VEGF/VEGFR2-targeted drugs.

• Bioassays and binding studies: Recombinant VEGFR2 is used in various bioassays, including surface plasmon resonance, cell proliferation assays, and ligand-binding studies, to quantify VEGF activity, screen inhibitors, and characterize molecular interactions.

• Lymphatic vessel research: Soluble forms of VEGFR2, including recombinant versions, are instrumental in studying lymphangiogenesis, as they can block VEGF-C-mediated proliferation and lymphatic vessel formation, which is relevant for cancer metastasis and tissue repair.

• Therapeutic development: Recombinant VEGFR2 serves as a model for developing and testing new biologics, such as neutralizing antibodies, fusion proteins, and small-molecule inhibitors targeting the VEGF/VEGFR2 axis.

Additional considerations:

• Recombinant VEGFR2 is available in various formats (e.g., Fc chimeras, monomeric/dimeric forms), each suited for specific experimental needs such as enhanced stability, avidity, or detection.
• It is commonly used in both in vitro (cell culture, biochemical assays) and in vivo (animal models) systems.

In summary, using recombinant human VEGF R2 allows for precise experimental control over VEGF signaling, enabling detailed mechanistic studies and the development of anti-angiogenic strategies in diverse biomedical research contexts.

Yes, recombinant human VEGF R2 can be used as a standard for quantification or calibration in ELISA assays, provided it is validated for this purpose and matches the assay’s requirements. Recombinant VEGF R2 is commonly used as a calibrator in quantitative sandwich ELISA kits designed to measure human VEGF R2 concentrations in biological samples.

Key technical considerations:

• Assay Compatibility: The ELISA kit must be validated to recognize both recombinant and natural forms of VEGF R2. Most commercial kits use recombinant human VEGF R2 as the standard and have demonstrated parallelism between recombinant and endogenous VEGF R2, indicating suitability for quantification.
• Standard Curve Preparation: Prepare a serial dilution of the recombinant VEGF R2 in the recommended calibrator diluent to generate a standard curve covering the assay’s dynamic range. Typical concentrations range from low picogram to nanogram levels, depending on kit sensitivity.
• Validation: Ensure the recombinant standard is of high purity and its concentration is accurately determined. The standard curve should be linear within the assay’s working range, and recovery and parallelism should be confirmed for your sample matrix.
• Limitations: Use the recombinant standard only for research purposes, not for diagnostic applications, unless specifically validated for clinical use. Do not mix standards or reagents from different lots or manufacturers, as this may affect quantification accuracy.

Best practices:

• Run a standard curve with each assay to ensure accurate quantification.
• Confirm that your recombinant VEGF R2 standard is compatible with the antibodies used in your ELISA (i.e., it contains the relevant epitopes recognized by the capture and detection antibodies).
• Validate recovery and linearity in your specific sample type (e.g., serum, plasma, cell lysate) to ensure accurate quantification.

Summary Table: Use of Recombinant VEGF R2 as ELISA Standard

In conclusion: Recombinant human VEGF R2 is suitable as a standard for ELISA quantification if it is validated for your assay and sample type, and you follow best practices for calibration and quality control.

Recombinant Human VEGF R2 (VEGFR2/KDR) has been validated in published research for a range of applications, primarily focused on studying VEGF signaling, angiogenesis, and therapeutic antibody development.

Key validated applications include:

• Bioassays: Used to assess the biological activity of VEGF and anti-VEGF agents, including neutralizing antibodies and engineered proteins targeting VEGFR2. These assays measure inhibition of VEGF-induced signaling, cell proliferation, and angiogenesis in vitro and in vivo.
• ELISA (Enzyme-Linked Immunosorbent Assay): Utilized as a standard or capture reagent to quantify VEGF or anti-VEGFR2 antibodies in biological samples.
• Surface Plasmon Resonance (SPR): Employed to characterize binding kinetics and affinities between VEGFR2 and its ligands or therapeutic antibodies.
• Binding Assays: Used to study the interaction between VEGFR2 and VEGF or other binding partners, including competitive binding and receptor-ligand specificity studies.
• Molecular Imaging: Applied in the development of imaging probes for visualizing VEGFR2 expression in angiogenic vasculature.
• Development and validation of therapeutic antibodies: Recombinant VEGFR2 is used as an antigen for screening, affinity maturation, and functional validation of monoclonal antibodies and bispecific constructs targeting VEGFR2 for anti-angiogenic therapy.
• Animal model validation: Recombinant human VEGFR2 has been used to generate and validate humanized mouse models for in vivo evaluation of anti-VEGFR2 therapeutics.

Supporting details:

• In published studies, recombinant VEGFR2 has been used to demonstrate the efficacy of novel antibodies (e.g., TTAC-0001, VEGFR-HK19) in inhibiting VEGF-induced angiogenesis and tumor growth.
• It is frequently used in cell-based assays to measure downstream signaling events, such as endothelial cell proliferation, migration, and survival, as well as in mechanistic studies of VEGF-VEGFR2 pathway modulation.
• Recombinant VEGFR2 is also a standard tool in the development of diagnostic and research assays for quantifying VEGF or anti-VEGFR2 agents in preclinical and translational research.

Summary of validated applications in published research:

These applications are well-documented in the literature and are central to angiogenesis research, drug development, and mechanistic studies of VEGF signaling.

To reconstitute and prepare Recombinant Human VEGF R2 (VEGFR2/KDR) protein for cell culture experiments, follow these best-practice steps based on current protocols and manufacturer recommendations:

1. Equilibrate and Centrifuge:

• Allow the lyophilized protein vial and your reconstitution buffer to reach room temperature.
• Briefly centrifuge the vial to ensure all powder is at the bottom.

2. Reconstitution Buffer:

• Most protocols recommend sterile PBS (phosphate-buffered saline) as the reconstitution buffer.
• For some preparations, sterile water can be used, but PBS is preferred for stability and physiological compatibility.
• If the protein is supplied with carrier protein (e.g., BSA), reconstitute in PBS containing at least 0.1% human or bovine serum albumin to minimize adsorption and stabilize the protein.

3. Reconstitution Concentration:

• Common reconstitution concentrations are 100 μg/mL or 1 mg/mL, depending on the specific product and intended use.
• Example: For a 100 μg vial, add 1 mL of buffer to achieve 100 μg/mL.

4. Dissolving the Protein:

• Add the appropriate volume of buffer directly to the vial.
• Gently swirl or invert the vial; avoid vigorous vortexing to prevent protein denaturation.
• Allow the protein to dissolve for 15–30 minutes at room temperature with gentle agitation. If undissolved material remains, continue gentle mixing for up to 2 hours.

5. Aliquoting and Storage:

• Once fully dissolved, aliquot the solution into single-use portions to avoid repeated freeze-thaw cycles.
• Store aliquots at –20°C to –70°C for long-term storage, or at 2–8°C for up to 1 month if used soon.
• Avoid repeated freeze-thaw cycles, as this can degrade the protein.

6. Preparation for Cell Culture:

• Before adding to cell culture, dilute the reconstituted stock to the desired working concentration using cell culture medium or PBS.
• If using in functional assays, ensure the final buffer is compatible with your cells (e.g., serum-free or serum-containing medium as appropriate).

Summary Table: VEGF R2 Protein Reconstitution

Additional Notes:

• Always consult the specific product datasheet for any unique requirements.
• If the protein is to be used in sensitive bioassays, consider adding carrier protein (BSA or HSA) to prevent loss due to adsorption.
• Use aseptic technique throughout to maintain sterility for cell culture applications.

These steps ensure optimal solubility, stability, and biological activity of recombinant VEGF R2 protein for cell-based experiments.