Recombinant Human VEGF₁₂₁ (a.a. 207-327)

Recombinant Human VEGF₁₂₁ (a.a. 207-327) is widely used in research applications due to its potent ability to stimulate angiogenesis, endothelial cell proliferation, migration, and survival, making it essential for studies involving vascular biology, wound healing, cancer, and tissue regeneration.

Key scientific reasons to use VEGF₁₂₁ (a.a. 207-327):

• Angiogenesis and Vasculogenesis: VEGF₁₂₁ is a major isoform of VEGF-A that induces the formation of new blood vessels (angiogenesis) and the development of vascular structures (vasculogenesis) in vitro and in vivo. This is critical for research on tumor growth, tissue engineering, and regenerative medicine.

• Endothelial Cell Function: It promotes proliferation, migration, and tube formation of endothelial cells, which are key steps in the formation of capillary networks. These properties are routinely exploited in assays such as HUVEC tube formation, wound healing, and cell migration studies.

• Wound Healing: VEGF₁₂₁ accelerates wound closure by enhancing endothelial cell migration and proliferation, making it valuable for studies on tissue repair and regeneration.

• Neuroprotection and Cell Survival: It supports the growth and survival of not only vascular endothelial cells but also dopaminergic neurons and astrocytes, broadening its utility to neurobiology research.

• Pathological Angiogenesis: VEGF₁₂₁ is involved in tumor angiogenesis and vascular leakage, making it a target and tool for cancer research, especially in evaluating anti-angiogenic therapies.

• Bioassay Standardization: Recombinant VEGF₁₂₁ (a.a. 207-327) is used as a standard in bioassays, ELISA capture, and functional studies due to its well-characterized activity and reproducibility.

• Isoform Specificity: The 121-amino acid isoform lacks heparin-binding domains present in longer VEGF-A isoforms, resulting in greater solubility and diffusibility, which is advantageous for certain experimental setups where localized or systemic effects are studied.

Typical applications include:

• Angiogenesis assays (e.g., tube formation, CAM assay)
• Endothelial cell proliferation and migration studies
• Wound healing models
• Cancer research (tumor vascularization)
• Neuroprotection and cell survival assays
• Standardization in ELISA and other bioassays

Summary:
Use recombinant human VEGF₁₂₁ (a.a. 207-327) in your research to reliably induce and study angiogenesis, endothelial cell biology, wound healing, and related processes, with broad utility in cancer, regenerative medicine, and neurobiology.

Yes, recombinant human VEGF₁₂₁ (a.a. 207-327) can be used as a standard for ELISA quantification, though there are important considerations regarding its suitability for your specific application.

Suitability as an ELISA Standard

The VEGF₁₂₁ isoform is a valid choice for ELISA calibration, particularly when your assay is specifically designed to detect this isoform. However, it's important to note that most commercial ELISA kits are calibrated against the VEGF₁₆₅ isoform (a.a. 27-191), which is the most abundant and potent VEGF-A variant. If you're using a standard VEGF ELISA kit, the calibration curve will have been optimized for VEGF₁₆₅, and using VEGF₁₂₁ as your standard may not provide accurate quantification of total VEGF in your samples.

Key Technical Considerations

Formulation Selection: The choice between carrier-free and BSA-containing formulations is critical for your application. For ELISA standard use, the formulation with bovine serum albumin (BSA) is generally recommended, as the carrier protein enhances stability and extends shelf-life. Conversely, if BSA could interfere with your assay—such as in certain bioassay applications or when using BSA-based blocking buffers—the carrier-free version is more appropriate.

Protein Characteristics: VEGF₁₂₁ exists as a disulfide-linked homodimeric protein with a molecular mass of approximately 28.4 kDa (consisting of two 121 amino acid chains). This isoform is unique among VEGF-A variants because it lacks a heparin-binding region and is freely diffusible, which may affect its behavior in certain assay formats compared to other isoforms.

Quality Parameters: When selecting your standard, verify that the preparation meets appropriate specifications for purity (≥95% by SDS-PAGE), endotoxin levels (≤0.1 EU/µg), and biological activity. These parameters ensure reliable quantification across your assay runs.

Practical Recommendations

Generate a fresh standard curve for each assay run rather than relying on previously established curves. Reconstitute the lyophilized protein in sterile water according to manufacturer specifications, and avoid repeated freeze-thaw cycles to maintain protein integrity. If your ELISA kit is calibrated against VEGF₁₆₅, consider whether you need to measure total VEGF or specifically the VEGF₁₂₁ isoform, as this will determine whether this standard is appropriate for your quantification goals.

Recombinant Human VEGF₁₂₁ (a.a. 207-327) has been validated for a range of applications in published research, primarily focused on its biological activity in angiogenesis, cell signaling, and as a standard or reagent in various assays.

Validated Applications in Published Research:

• Bioassays:
  VEGF₁₂₁ (a.a. 207-327) is widely used in bioassays to assess its ability to stimulate angiogenesis, endothelial cell proliferation, migration, and tube formation. These assays are typically performed using human umbilical vein endothelial cells (HUVECs) and other endothelial cell types.

  • Examples include:
    • Induction of tube formation and branching in HUVECs (in vitro angiogenesis assays).
    • Promotion of endothelial cell proliferation and migration.
    • Stimulation of angiogenesis in the chicken chorioallantoic membrane (CAM) assay.
    • Functional studies of VEGF signaling in various cell lines.

• ELISA (Enzyme-Linked Immunosorbent Assay):
  Used as a standard or capture reagent in ELISA to quantify VEGF levels in biological samples or to validate antibody specificity.

• Western Blot:
  Applied as a positive control or to validate the presence and purity of VEGF₁₂₁ in recombinant preparations and cell lysates.

• Surface Plasmon Resonance (SPR):
  Utilized to study protein-protein interactions, such as binding to receptors or extracellular matrix components.

• In vivo Angiogenesis Models:
  Used in animal models (e.g., CAM assay, mouse models) to evaluate proangiogenic activity and therapeutic potential in wound healing and tumor angiogenesis.

• Cell Culture Supplement:
  Added to cell culture media to promote the growth and survival of endothelial cells, dopaminergic neurons, and astrocytes.

• Mechanistic and Signaling Studies:
  Employed to dissect VEGF receptor signaling pathways, alternative splicing regulation, and resistance mechanisms to anti-VEGF therapies.

Summary Table of Key Validated Applications

Key References Supporting These Applications:

• PLoS ONE, 2022; Cell Reports, 2021; Sci Rep, 2019; FASEB J, 2010; Anticancer Res, 2014; PMC4137420 (tube formation, proliferation); PMC7049456 (CAM assay, migration, wound healing).

Additional Context:

• VEGF₁₂₁ (a.a. 207-327) is a major isoform of VEGF-A, lacking the heparin-binding domain, making it highly diffusible and suitable for studies where paracrine signaling is of interest.
• It is commonly used in research on angiogenesis, cancer, vascular biology, and regenerative medicine.

If you need protocol details or specific assay recommendations, please specify the intended application.

To reconstitute and prepare Recombinant Human VEGF₁₂₁ (a.a. 207-327) for cell culture experiments, dissolve the lyophilized protein in sterile 4 mM HCl to a final concentration of 100 μg/mL. After reconstitution, further dilute the stock solution in cell culture medium or buffer containing a carrier protein such as 0.1% BSA or HSA in PBS to minimize adsorption and enhance stability.

Essential steps and considerations:

• Reconstitution:

  • Add sterile 4 mM HCl directly to the lyophilized VEGF₁₂₁ protein to achieve 100 μg/mL.
  • Gently mix by pipetting; avoid vigorous vortexing to prevent protein denaturation.
  • If the product contains BSA as a carrier, follow the same protocol; if carrier-free, consider adding BSA or HSA during dilution.

• Aliquoting and Storage:

  • Aliquot the reconstituted stock to avoid repeated freeze-thaw cycles.
  • Store aliquots at −20 °C to −70 °C for long-term stability.
  • For short-term use (1–2 weeks), storage at +4 °C is acceptable.

• Working Solution Preparation:

  • Dilute the stock solution into cell culture medium or PBS containing 0.1% BSA or HSA to reach the desired final concentration for your assay (e.g., 10–500 ng/mL for endothelial cell assays).
  • Prepare working solutions fresh before use.

• Sterility:

  • Ensure all buffers and diluents are sterile and endotoxin-free, as VEGF₁₂₁ is highly sensitive to contamination, which can affect cell culture results.

• Application:

  • Typical concentrations for cell culture experiments range from 10–500 ng/mL, depending on cell type and assay (e.g., HUVEC proliferation, migration, or tube formation assays).

Summary Table: Preparation Protocol

Best Practices:

• Always consult the specific product datasheet for any manufacturer-specific recommendations regarding buffer composition and storage.
• Use low-protein binding tubes and pipette tips to minimize loss due to adsorption.
• Confirm protein activity with a pilot experiment (e.g., HUVEC proliferation or migration assay) before large-scale use.

If your experiment requires a different buffer (e.g., PBS), ensure the protein is fully soluble and stable in that buffer by performing a small-scale test. Avoid high ionic strength or extreme pH conditions unless validated for VEGF₁₂₁ stability.