Recombinant Human VEGF 165 Protein

Recombinant Human VEGF 165 Protein is widely used in research because it is a key regulator of angiogenesis (formation of new blood vessels), vasculogenesis, and endothelial cell function, making it essential for studies in vascular biology, cancer, regenerative medicine, and stem cell differentiation.

Key reasons to use recombinant human VEGF 165 in research applications:

• Angiogenesis and Vascular Biology: VEGF 165 is the most biologically active isoform of VEGF-A, directly stimulating proliferation, migration, and survival of endothelial cells, which are critical for blood vessel formation in both physiological (e.g., wound healing, tissue regeneration) and pathological (e.g., tumor growth, diabetic retinopathy) contexts.
• Stem Cell Differentiation: It is commonly added to differentiation protocols to guide pluripotent stem cells toward endothelial or hematopoietic lineages, and to support the formation of organoids such as kidney podocytes and liver spheres.
• Functional Assays: VEGF 165 is used in bioassays to test endothelial cell proliferation (e.g., HUVEC assays), migration, and tube formation, serving as a positive control or experimental variable in angiogenesis models.
• Cancer Research: Because tumors exploit VEGF-driven angiogenesis for growth and metastasis, VEGF 165 is used to model tumor microenvironments, study anti-angiogenic therapies, and investigate mechanisms of tumor vascularization.
• Regenerative Medicine: VEGF 165 supports vascularization in engineered tissues and enhances bone and tissue repair by promoting blood vessel growth, which is crucial for tissue survival and integration.
• Neurobiology: It has neurotrophic effects, promoting the growth and survival of neurons and astrocytes, and plays a role in neurogenesis.
• Immunology and Inflammation: VEGF 165 can act as a chemoattractant for monocytes and osteoblasts, and modulate inflammatory responses, making it relevant in studies of immune cell migration and inflammatory diseases.

Technical advantages of recombinant protein:

• Consistency and Purity: Recombinant production ensures high purity and batch-to-batch consistency, which is critical for reproducible results in sensitive assays.
• Defined Activity: Biological activity is well-characterized, allowing precise dosing and reliable interpretation of experimental outcomes.
• Animal-Free Production: Many recombinant VEGF 165 preparations are produced without animal-derived components, reducing variability and risk of contamination.

Summary of applications:

• Endothelial cell proliferation and migration assays
• Angiogenesis and tube formation assays
• Stem cell differentiation protocols
• Tumor angiogenesis models
• Tissue engineering and regenerative medicine studies
• Neurogenesis and neuroprotection assays
• Inflammatory and immune cell migration studies

Using recombinant human VEGF 165 protein provides a reliable, well-characterized tool for dissecting the molecular mechanisms of angiogenesis and related biological processes across a wide range of research fields.

You can use recombinant human VEGF 165 protein as a standard for quantification or calibration in ELISA assays, provided the protein is of high purity, its concentration is accurately known, and it is compatible with your assay system.

Key considerations and supporting details:

• ELISA Calibration: Many commercial human VEGF ELISA kits are calibrated using highly purified recombinant human VEGF 165 protein, often expressed in systems such as Sf21 insect cells or E. coli. These kits use recombinant VEGF 165 as the reference standard to generate the standard curve for quantification.
• Assay Compatibility: The recombinant VEGF 165 protein must be recognized by the capture and detection antibodies used in your ELISA. Most sandwich ELISAs for VEGF 165 are designed to detect both natural and recombinant forms, as confirmed by multiple kit inserts.
• Reference Standards: Some recombinant VEGF 165 proteins are calibrated against international standards, such as the WHO/NIBSC 02/286 reference reagent. If your recombinant protein is traceable to such a standard, it increases the reliability and comparability of your quantification.
• Concentration and Purity: Ensure the recombinant VEGF 165 protein you use has a well-characterized concentration (e.g., determined by absorbance at 280 nm or amino acid analysis) and is free from contaminants that could interfere with the assay.
• Preparation: Prepare a dilution series of the recombinant VEGF 165 protein in the same buffer or matrix as your samples to generate a standard curve. This allows accurate interpolation of sample concentrations from the curve.

Best Practices:

• Use the same diluent for standards and samples to minimize matrix effects.
• If possible, use a recombinant VEGF 165 standard that is calibrated against the WHO reference reagent for greater accuracy and comparability.
• Validate that your ELISA detects the recombinant standard with similar efficiency as endogenous VEGF 165, especially if your samples are complex biological fluids.

Summary Table: Use of Recombinant VEGF 165 as ELISA Standard

In summary, recombinant human VEGF 165 protein is widely used and accepted as a standard for ELISA quantification, provided it meets the above criteria. Always verify compatibility with your specific assay system.

Recombinant Human VEGF 165 Protein has been validated for a broad range of applications in published research, primarily related to angiogenesis, cell signaling, and stem cell biology.

Key validated applications include:

• Functional Assays: Used to assess biological activity, such as stimulation of endothelial cell proliferation, migration, and tube formation, which are hallmark assays for angiogenesis.
• ELISA (Enzyme-Linked Immunosorbent Assay): Utilized as a standard or analyte to quantify VEGF levels in biological samples.
• Western Blot: Applied for detection and quantification of VEGF or its downstream signaling components in cell and tissue lysates.
• Immunohistochemistry: Used to visualize VEGF expression and localization in tissue sections.
• Immunoprecipitation: Employed to isolate VEGF or its interacting partners from complex mixtures.
• Bioassays: Extensively used in published studies to stimulate or modulate cellular responses, including:
  • Endothelial cell proliferation and migration.
  • Differentiation of stem cells into hematopoietic progenitors, kidney podocytes, or liver spheres.
  • Support of angiogenesis in stem cell transplantation models.
  • In vivo assays for tumor growth, vascularization, and anti-angiogenic therapy in animal models.
  • Stimulation of spheroid cultures and whole tissue explants for cancer and vascular biology research.
• Apoptosis Assays: Used to study the effect of VEGF on cell survival and apoptosis.
• 3D Cell Culture: Applied in organoid and spheroid models to study angiogenesis and tumor biology.
• Cell and Gene Therapy Research: Used to enhance vascularization and cell migration in regenerative medicine protocols.

Additional validated uses in published literature:

• Anti-cancer research: Recombinant VEGF 165b isoform has been shown to inhibit tumor growth and vessel density in vivo, indicating therapeutic potential as an anti-angiogenic agent.
• Stem cell differentiation protocols: VEGF 165 is a critical growth factor for directing pluripotent stem cells toward hematopoietic, renal, and hepatic lineages.
• Vascular biology: Used to study mechanisms of vasculogenesis, angiogenesis, and vascular permeability.

In summary, Recombinant Human VEGF 165 Protein is a well-validated reagent for functional, biochemical, and cell-based assays in angiogenesis, cancer, stem cell, and regenerative medicine research, with extensive documentation in peer-reviewed publications.

To reconstitute and prepare Recombinant Human VEGF 165 Protein for cell culture experiments, dissolve the lyophilized protein in an appropriate sterile buffer, typically at a concentration of 100–500 μg/mL, using either sterile 4 mM HCl (with or without 0.1% serum albumin) or sterile deionized water, depending on the formulation and manufacturer’s instructions.

General reconstitution protocol:

• Carrier-free formulations:
  Reconstitute in sterile 4 mM HCl at 100–500 μg/mL.
  For some products, sterile deionized water may be used at 200–500 μg/mL.

• Formulations with carrier protein (e.g., BSA):
  Reconstitute in sterile 4 mM HCl containing 0.1% human or bovine serum albumin at 100–500 μg/mL.
  Alternatively, some protocols recommend sterile PBS (pH 7.4) with 0.1% recombinant serum albumin at 0.2 mg/mL.

Best practices for reconstitution:

• Briefly centrifuge the vial to collect all lyophilized powder at the bottom before opening.
• Add the recommended volume of sterile buffer gently down the side of the vial; do not vortex.
• Mix by gentle swirling or tapping until fully dissolved.
• Allow the protein to dissolve for several minutes at room temperature.
• If necessary, further dilute the stock solution in cell culture medium or buffer immediately before use.

Storage after reconstitution:

• Store the reconstituted protein at 2–8 °C for up to one month, or at −20 °C to −70 °C for longer periods.
• Avoid repeated freeze-thaw cycles to preserve protein activity.

Application in cell culture:

• Typical working concentrations for stimulation of endothelial cells (e.g., HUVECs) range from 1.5–12 ng/mL.
• Prepare working dilutions in cell culture medium immediately before use.

Critical notes:

• Always consult the Certificate of Analysis (CoA) or product datasheet for specific instructions, as formulations and recommended buffers may vary.
• Use endotoxin-free reagents and sterile technique throughout to prevent contamination and ensure reproducibility.

Summary Table:

Always verify the recommended buffer and concentration for your specific VEGF 165 product before use.