Recombinant Rat VEGF₁₆₄

Recombinant Rat VEGF₁₆₄ is a highly valuable tool for research applications due to its well-characterized biological activity and relevance in multiple physiological and pathological processes. Here are several key reasons why you should consider using Recombinant Rat VEGF₁₆₄ in your studies:

1. Critical Role in Angiogenesis

VEGF₁₆₄ is a major isoform of vascular endothelial growth factor (VEGF) in rats and plays a central role in promoting angiogenesis—the formation of new blood vessels. This makes it essential for studies involving vascular development, wound healing, tissue engineering, and ischemic disease models.

2. Stimulates Endothelial Cell Proliferation and Survival

Recombinant Rat VEGF₁₆₄ potently stimulates the proliferation, survival, migration, and permeability of endothelial cells in a dose-dependent manner. This activity is crucial for in vitro assays and in vivo studies where endothelial cell function is being investigated.

3. Species-Specific Relevance

Using rat-specific VEGF₁₆₄ ensures compatibility with rat models, which are widely used in preclinical research. This is particularly important for studies involving rat cell lines, primary cells, or in vivo rat models, as it avoids potential cross-species reactivity issues.

4. Well-Characterized Bioactivity

Recombinant Rat VEGF₁₆₄ has been extensively validated in functional assays. For example, it stimulates proliferation in human umbilical vein endothelial cells (HUVECs) with a well-defined ED₅₀ (effective dose for 50% response), allowing for precise dosing and reproducible results.

5. Multiple Applications

• In vitro bioassays: Study endothelial cell responses, signaling pathways, and drug effects.
• In vivo studies: Investigate angiogenesis, vascular permeability, and tissue regeneration in rat models.
• Tissue engineering: Promote vascularization of engineered tissues or implants.
• Disease modeling: Explore the role of VEGF in cancer, diabetic retinopathy, ischemic diseases, and inflammatory conditions.

6. High Purity and Consistency

Commercially available recombinant Rat VEGF₁₆₄ is produced under controlled conditions, ensuring high purity, batch-to-batch consistency, and minimal contamination, which is critical for reliable and reproducible experimental results.

7. Relevance to Pathological and Physiological Processes

VEGF₁₆₄ is involved in both physiological (e.g., development, wound healing) and pathological (e.g., tumor angiogenesis, diabetic retinopathy) processes. Using the recombinant protein allows researchers to dissect its specific contributions in these contexts.

8. Compatibility with Rat Models

Since rat VEGF₁₆₄ shares high sequence homology with mouse and human VEGF, findings from rat studies can often be extrapolated to other species, enhancing the translational relevance of your research.

In summary, Recombinant Rat VEGF₁₆₄ is a powerful and versatile reagent for studying angiogenesis, endothelial cell biology, and vascular-related diseases in rat models. Its well-defined activity, species specificity, and broad applicability make it an excellent choice for both basic and applied research.

Yes, recombinant rat VEGF₁₆₄ can be used as a standard for quantification and calibration in ELISA assays, though there are important considerations regarding its application and formulation.

Suitability as an ELISA Standard

Recombinant rat VEGF₁₆₄ is specifically designed and validated for use as an ELISA standard. The protein demonstrates excellent characteristics for this purpose, including high purity (greater than 95% as determined by reducing SDS-PAGE) and low endotoxin levels (less than 0.1 ng/µg). Multiple ELISA kit formulations utilize recombinant rat VEGF₁₆₄ as their calibration standard.

Biological Activity and Functional Validation

The recombinant protein exhibits robust biological activity, which is critical for accurate quantification. When immobilized at 2 μg/mL, rat VEGF₁₆₄ effectively binds to VEGF receptors with well-characterized binding kinetics. In functional ELISA assays, the protein demonstrates a linear binding range of 0.03-3.6 ng/mL when interacting with human KDR receptors. Additionally, the protein stimulates cell proliferation in human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner, with ED₅₀ values typically ranging from 0.02-0.10 ng/mL to 0.75-3.75 ng/mL depending on the specific assay conditions.

Formulation Considerations

Carrier protein selection is an important factor when preparing your standard. For ELISA applications, recombinant rat VEGF₁₆₄ is recommended in formulations containing bovine serum albumin (BSA) or other carrier proteins. This formulation choice is particularly important for maintaining protein stability and preventing non-specific binding during assay procedures.

Preparation and Storage Guidelines

When reconstituting the lyophilized protein for use as a standard, follow these best practices:

• Dissolve in distilled water without vortexing or vigorous pipetting
• Avoid reconstituting to concentrations below 100 μg/mL
• Aliquot the reconstituted solution to minimize freeze-thaw cycles
• Store reconstituted protein at 4-7°C for 2-7 days, or at temperatures below -20°C for up to 3 months

Important Limitation

One critical caveat: ELISA standards are typically not recommended for bioassay applications, as they are not validated for stimulation or functional assays. If your intended use involves assessing biological activity rather than simple quantification, you should verify that your specific recombinant preparation has been tested for bioassay applications.

Recombinant Rat VEGF₁₆₄ has been validated in published research for a range of applications, primarily related to its roles in angiogenesis, vascular permeability, and neurovascular biology in rat models.

Key validated applications include:

• In vivo angiogenesis assays: Used to stimulate and assess blood vessel formation in various rat tissues and disease models, such as tissue-engineered constructs, spinal cord injury, and tumor angiogenesis.
• Bioassays with whole cells: Applied to evaluate the angiogenic and proliferative responses of mesenchymal stem cells, endothelial cells, and other cell types in vitro and ex vivo.
• Stimulation of cell cultures: Used to induce angiogenic signaling pathways, endothelial cell migration, and proliferation in cell culture supernatants and tissue explants.
• In vivo models of disease: Employed in studies of diabetic retinopathy, choroidal neovascularization (CNV), and tissue regeneration to model pathological and therapeutic angiogenesis.
• Functional characterization of anti-angiogenic therapeutics: Used as a positive control or stimulant in the evaluation of anti-VEGF drugs and inhibitors in vivo and in vitro.
• Assessment of vascular permeability and inflammation: Demonstrated to induce blood-retinal barrier breakdown and leukostasis in diabetic retina models, highlighting its proinflammatory properties.
• Osteogenesis and tissue engineering: Combined with biomaterials and physical stimuli (e.g., electromagnetic fields) to promote bone and vascular tissue regeneration in cranial defect models.

Representative published research applications:

• Neurovascular protection: Sustained release from nanofiber membranes reduced oxygen/glucose deprivation-induced injury to neurovascular units in rats.
• Bone regeneration: Promoted osteogenesis and angiogenesis in mesenchymal stem cell-laden implants in rat cranial defect models.
• Tumor angiogenesis: Used to study the modulation of angiogenesis and tumor growth by dietary compounds and gene therapy approaches.
• Retinal disease models: Validated for inducing and studying retinal neovascularization and blood-retinal barrier breakdown in diabetic and CNV models.
• Tissue engineering: Applied in the vascularization of engineered tissues, such as intestine and esophagus, in rat transplantation models.

Summary of sample types and experimental contexts:

• In vivo (whole animal, tissue, and organ models)
• Whole cells (primary cultures, stem cells, endothelial cells)
• Cell culture supernatants (for stimulation and signaling studies)

These applications are supported by multiple peer-reviewed studies and are widely used in angiogenesis, tissue engineering, and disease modeling research involving rats.

To reconstitute and prepare Recombinant Rat VEGF₁₆₄ protein for cell culture experiments, follow these steps to ensure protein stability and biological activity:

1. Centrifuge the vial briefly before opening to collect all lyophilized material at the bottom.

2. Reconstitution buffer:

   • Use sterile distilled water or sterile PBS (phosphate-buffered saline).
   • For enhanced stability and to prevent adsorption, add a carrier protein such as 0.1%–1% BSA (bovine serum albumin) or HSA (human serum albumin).

3. Concentration:

   • Typical stock concentrations range from 0.1–1.0 mg/mL.
   • Commonly, 100–250 μg/mL is used for initial reconstitution.

4. Dissolving the protein:

   • Gently add the buffer to the vial.
   • Do not vortex or pipette vigorously; instead, gently mix by slow inversion or gentle pipetting to avoid protein denaturation.

5. Aliquoting:

   • Once fully dissolved, divide the solution into small aliquots to avoid repeated freeze-thaw cycles.
   • Store aliquots at ≤ –20°C for long-term storage; short-term storage (2–7 days) can be at 4–8°C.

6. Working solution preparation:

   • For cell culture, further dilute the stock solution in culture medium or buffer containing FBS or tissue culture grade BSA to minimize protein loss and maintain low endotoxin levels.
   • The final working concentration should be optimized for your specific assay; published ED₅₀ values for endothelial cell proliferation are typically 0.75–3.75 ng/mL.

Summary Table:

Additional Notes:

• Always use low endotoxin reagents and buffers for cell culture applications.
• Avoid repeated freeze-thaw cycles to preserve protein activity.
• If long-term storage is needed, consider adding stabilizers such as 5% HSA, 10% FBS, or 5% trehalose.

These steps will help maintain the integrity and biological activity of recombinant rat VEGF₁₆₄ for reliable cell culture experiments.