Recombinant Human VEGF-D

Recombinant Human VEGF-D is used in research applications to study and manipulate processes involving angiogenesis (formation of new blood vessels), lymphangiogenesis (formation of lymphatic vessels), and endothelial cell biology.

Key scientific reasons to use recombinant VEGF-D include:

• Modeling Angiogenesis and Lymphangiogenesis: VEGF-D is a potent stimulator of both blood and lymphatic vessel growth. It binds primarily to VEGFR-2 and VEGFR-3 receptors on endothelial cells, promoting their proliferation, migration, and survival. This makes it valuable for in vitro assays (e.g., tube formation, migration, proliferation) and in vivo models of vascular development, tissue regeneration, and wound healing.

• Disease Mechanism Studies: VEGF-D is implicated in the progression of various diseases, including cancer (tumor angiogenesis and metastasis), cardiovascular conditions (e.g., refractory angina, peripheral vascular disease), and pulmonary disorders (e.g., lymphangioleiomyomatosis, pulmonary edema). Recombinant VEGF-D enables mechanistic studies and therapeutic target validation in these contexts.

• Therapeutic Development: Recombinant VEGF-D is used to evaluate its potential as a therapeutic agent for promoting vascular or lymphatic regeneration, such as in lymphedema, tissue ischemia, or after lymph node transplantation. It also serves as a tool for testing anti-angiogenic or anti-lymphangiogenic drugs.

• Functional and Structural Analysis: Recombinant forms allow precise control over protein concentration and structure, facilitating studies on receptor binding, signaling pathways, and structure-function relationships.

• Diagnostics and Biomarker Research: VEGF-D levels are used as biomarkers in certain diseases (e.g., lymphangioleiomyomatosis), and recombinant protein is essential for assay development and validation.

Typical applications include:

• Endothelial cell proliferation, migration, and tube formation assays
• Animal models of angiogenesis, lymphangiogenesis, and tissue repair
• Mechanistic studies of VEGF-D/VEGFR signaling
• Preclinical evaluation of pro- or anti-angiogenic therapies
• Development of diagnostic assays for VEGF-D quantification

Best practices: Use recombinant VEGF-D at concentrations validated for your cell type or model system, and include appropriate controls (e.g., vehicle, other VEGF family members) to distinguish specific effects.

In summary, recombinant human VEGF-D is a versatile tool for investigating vascular and lymphatic biology, disease mechanisms, and therapeutic strategies in both basic and translational research.

Recombinant human VEGF-D can be used as a standard for quantification or calibration in ELISA assays, provided it is well-characterized and matches the form of VEGF-D detected by your assay. This is a common practice in quantitative ELISA protocols for growth factors and cytokines.

Key considerations and supporting details:

• ELISA kits for VEGF-D typically use recombinant human VEGF-D as the standard. For example, the Quantikine Human VEGF-D ELISA kit uses Sf21-expressed recombinant human VEGF-D as its standard, and the standard curves generated with this recombinant protein are parallel to those obtained with naturally occurring VEGF-D in biological samples. This demonstrates that recombinant VEGF-D is suitable for calibration and quantification in these assays.

• Parallelism and recovery data confirm suitability. The referenced kit reports that results for natural VEGF-D are linear and parallel to the standard curve generated with recombinant VEGF-D, indicating that the recombinant protein is appropriate for quantification. Recovery experiments also show high accuracy when recombinant VEGF-D is spiked into various matrices.

• Assay specificity and form of VEGF-D. Ensure that the recombinant VEGF-D you use as a standard matches the isoform and post-translational processing state recognized by the antibodies in your ELISA. Most commercial recombinant VEGF-D is the mature, fully processed form, which is typically what ELISA antibodies are raised against. If your samples contain a different form (e.g., precursor or partially processed), quantification may be less accurate.

• Validation and documentation. When using recombinant VEGF-D from a source other than the kit manufacturer, validate that your standard curve is parallel to the kit’s standard curve and that recovery and linearity are acceptable in your sample matrix. This is especially important if you are using a custom or in-house ELISA protocol.

• Best practices:

  • Prepare a fresh standard curve for each assay run.
  • Use the same buffer and matrix for your standards as for your samples to minimize matrix effects.
  • Confirm that your recombinant VEGF-D is of high purity and correctly folded, as misfolded or aggregated protein may not be recognized by the ELISA antibodies.

In summary:
You can use recombinant human VEGF-D as a standard for ELISA quantification, provided it is well-characterized, matches the form detected by your assay, and you validate its performance in your specific protocol.

Recombinant Human VEGF-D has been validated for a range of applications in published research, primarily in studies of angiogenesis, lymphangiogenesis, cell signaling, and disease models. Key validated applications include:

• Cell-based bioassays: Used to assess endothelial cell proliferation, migration, and tube formation, particularly in human microvascular endothelial cells.
• Immunohistochemistry (IHC): Applied to tissue samples to detect VEGF-D expression and study its role in tumor lymphangiogenesis and resistance to anti-angiogenic therapies.
• Western blotting: Used to analyze VEGF-D protein levels and signaling pathway activation in cell lysates.
• ELISA (as control): Employed as a standard or control in quantitative assays measuring VEGF-D in serum or other biological fluids.
• Animal models: Delivered via protein injection or gene transfer to study therapeutic angiogenesis, lymphangiogenesis, and tissue regeneration in cardiovascular disease, lymphedema, inflammation, and wound healing.
• Alginate hydrogel release studies: Incorporated into biomaterials for sustained release and evaluation of lymphangiogenic therapeutic potential.
• Functional assays: Used to demonstrate the induction of new blood and lymphatic vessels, and to evaluate effects on tissue edema and lymphedema.

Additional validated uses include:

• Tumor biology research: Investigating VEGF-D’s role in cancer cell autocrine signaling, metastatic potential, and microvascular patterning.
• Disease biomarker studies: Assessing VEGF-D as a biomarker for disease progression or therapeutic response, especially in cancer and pulmonary diseases.
• Cardiovascular and ocular disease models: Evaluating therapeutic angiogenesis and vascular remodeling in preclinical and clinical studies.

These applications are supported by peer-reviewed publications and are widely used in both basic and translational research settings.

To reconstitute and prepare Recombinant Human VEGF-D protein for cell culture experiments, dissolve the lyophilized protein in sterile, high-purity water to a concentration of at least 100 µg/ml. Avoid vigorous mixing; gently swirl or invert the vial to ensure complete dissolution.

Step-by-step protocol:

• Equilibrate the lyophilized vial and the reconstitution water to room temperature before opening.
• Centrifuge the vial briefly (e.g., 6000–10,000 rpm for 30 s) to collect all powder at the bottom.
• Add sterile, high-purity water (e.g., 18 MΩ-cm) to achieve the desired concentration (commonly 100 µg/ml or as specified in your protocol).
• Gently mix by swirling or inverting; do not vortex, as this can denature the protein.
• Allow to dissolve for 15–30 minutes at room temperature. If undissolved material remains, continue gentle mixing for up to 2 hours.
• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
• Store aliquots at −20 °C or below for long-term storage (up to 3 months); for short-term use, store at 4–8 °C for up to 2–7 days.

Dilution for cell culture:

• Before adding to cell culture, dilute the stock solution in cell culture medium or buffer containing a carrier protein (e.g., 0.1% BSA or 1% serum) to prevent adsorption and loss of activity.
• Typical working concentrations for cell stimulation are in the range of 1–100 ng/ml, but optimal dosing should be determined empirically for your specific assay.

Additional notes:

• If the protein was lyophilized from PBS or another buffer, check the datasheet for any required buffer exchange or additional components (e.g., trehalose, mannitol, Tween 80).
• Avoid repeated freeze-thaw cycles, as this can degrade the protein.
• Always use sterile technique to prevent contamination.

Summary of best practices:

• Use sterile, high-purity water for initial reconstitution.
• Gently mix; do not vortex.
• Aliquot and store at −20 °C or below.
• Dilute in cell culture medium with carrier protein before use.

These steps ensure that VEGF-D retains its biological activity and is suitable for cell culture applications.