Recombinant Human PlGF-1

Recombinant Human PlGF-1 is widely used in research due to its critical role in angiogenesis, inflammation, tissue repair, and disease modeling, particularly through its interaction with the VEGFR-1/FLT1 receptor.

Key scientific applications and rationales for using Recombinant Human PlGF-1 include:

• Angiogenesis Research: PlGF-1 is a potent proangiogenic factor, enhancing endothelial cell proliferation, migration, and survival, making it essential for studying blood vessel formation in both physiological and pathological contexts such as wound healing, tumor growth, and ischemic diseases.
• Inflammation and Immune Modulation: PlGF-1 induces monocyte activation, migration, and the production of inflammatory cytokines, facilitating studies on immune cell recruitment and inflammatory responses.
• Disease Modeling: PlGF-1 is implicated in various diseases, including cancer (promoting tumor cell migration and angiogenesis), diabetic complications (such as retinal and macular edema), pre-eclampsia, and cardiovascular disorders. Its use allows for mechanistic studies and therapeutic target validation in these models.
• Tissue Repair and Regeneration: PlGF-1 supports wound and bone fracture healing by promoting angiogenesis and recruiting reparative cells, making it valuable in regenerative medicine research.
• Bioassays and ELISA Standards: Recombinant PlGF-1 is used as a standard or positive control in ELISA, binding assays, and cell-based bioassays to quantify or validate PlGF-related signaling pathways.
• Therapeutic Investigation: Administration of recombinant PlGF-1 has been shown to modulate blood pressure and vascular function in animal models of hypertension and pre-eclampsia, supporting its use in translational research for vascular disorders.

Mechanistically, PlGF-1 acts primarily through VEGFR-1/FLT1, influencing both direct endothelial responses and indirect effects on nonvascular cells, thus providing a versatile tool for dissecting complex signaling networks in angiogenesis and inflammation.

Summary of advantages:

• High specificity and activity for VEGFR-1/FLT1 signaling studies.
• Consistent, defined reagent for reproducible results in cell-based and biochemical assays.
• Enables modeling of human disease processes and evaluation of therapeutic interventions.

In summary, using Recombinant Human PlGF-1 in research provides a controlled, reliable means to investigate angiogenic signaling, immune modulation, tissue repair, and disease mechanisms relevant to human health and disease.

Yes, recombinant Human PlGF-1 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for this purpose. Recombinant PlGF-1 is commonly used as a standard in commercial ELISA kits designed to quantify human PlGF in biological samples.

Key considerations and supporting details:

• Parallelism and Accuracy: ELISA kits that use recombinant human PlGF-1 as a standard have demonstrated that dose-response curves for recombinant and natural PlGF are parallel, indicating that recombinant PlGF-1 is suitable for quantification of endogenous PlGF in samples. This parallelism is essential for accurate calibration and quantification.

• Validation: It is critical that the recombinant PlGF-1 standard is validated for use in your specific ELISA system. Commercial kits typically provide recombinant PlGF-1 standards that have been tested for accuracy, recovery, and precision in various sample matrices (serum, plasma, cell culture supernatant). If you are developing your own assay, you should confirm that your recombinant PlGF-1 behaves similarly to endogenous PlGF in your assay conditions.

• Isoform and Sequence: Ensure that the recombinant PlGF-1 matches the isoform and sequence recognized by your ELISA antibodies. Most kits specify the isoform and the amino acid sequence used for the standard.

• Carrier Proteins and Purity: For use as an ELISA standard, recombinant PlGF-1 should be of high purity and, ideally, carrier-free unless your assay specifically requires a carrier protein for stability.

• Bioactivity vs. Quantification: While recombinant PlGF-1 may be biologically active, for ELISA calibration, the critical factor is immunoreactivity and parallelism with the native protein, not bioactivity per se.

Best Practices:

• Reconstitute and store the recombinant standard according to the manufacturer’s instructions to maintain stability and reproducibility.
• Prepare a standard curve using serial dilutions of the recombinant PlGF-1 to calibrate your assay.
• Include appropriate controls and validate recovery and linearity in your sample matrix.

Limitations:

• If your ELISA is designed to detect only native PlGF or a specific isoform, confirm that the recombinant standard is appropriate for your assay.
• Recombinant proteins expressed in different systems (e.g., E. coli vs. mammalian cells) may have different post-translational modifications, which can affect antibody recognition in rare cases.

Summary Table: Use of Recombinant Human PlGF-1 as ELISA Standard

In conclusion, recombinant Human PlGF-1 is widely used and accepted as a standard for ELISA quantification, provided it is validated for your specific assay system.

Research Applications of Recombinant Human PlGF-1

Recombinant human PlGF-1 has been validated across multiple research applications in published literature, reflecting its importance as an angiogenic factor.

Bioassay and Functional Studies

The primary application domain involves bioassays examining PlGF-1's effects on endothelial and vascular cells. Published research has utilized recombinant PlGF-1 to investigate hypoxia-induced responses in endothelial colony-forming cells, demonstrating how this growth factor modulates cellular behavior under stress conditions. Additionally, bioassays have been employed to study PlGF-1's role in angiogenesis, including its involvement in plasminogen activator inhibitor-1 (PAI-1) induction in pathological contexts such as sickle cell disease.

Binding and Receptor Interaction Studies

Binding assays represent another validated application, particularly for examining PlGF-1 interactions with its cognate receptor VEGFR-1/FLT1. These assays have been instrumental in characterizing the molecular mechanisms by which PlGF-1 signals through this receptor to stimulate endothelial cell proliferation and migration.

Cardiovascular Development and Regeneration

Recent research has validated recombinant PlGF-1 protein in differentiation assays for cardiac and vascular tissue engineering. Treatment with recombinant PlGF-1 protein at concentrations of 50-100 ng/mL during specific differentiation windows improved the induction of cardiomyocytes, smooth muscle cells, and endothelial cells from human embryonic stem cell-derived progenitors. These studies confirmed both in vitro and in vivo cardiomyogenic and vasculogenic effects, demonstrating PlGF-1's therapeutic potential for cardiac muscle graft development.

Analytical and Biochemical Characterization

Recombinant PlGF-1 has been validated for use in analytical applications including HPLC, SDS-PAGE, and mass spectrometry, as well as in sandwich ELISA assays for quantification purposes. These applications support both protein characterization and biomarker measurement in clinical and research contexts.

To reconstitute and prepare Recombinant Human PlGF-1 protein for cell culture experiments, dissolve the lyophilized protein in a suitable sterile buffer, typically at a concentration of 0.1–1.0 mg/mL, then dilute to your working concentration in cell culture medium. The most common buffers are sterile distilled water, phosphate-buffered saline (PBS), or 0.1 M acetic acid, depending on the specific formulation and downstream application.

Step-by-step protocol:

• Equilibrate vial and buffer: Allow both the lyophilized protein vial and your chosen buffer to reach room temperature before opening to prevent condensation.
• Centrifuge vial: Briefly centrifuge the vial to collect the powder at the bottom before opening.
• Reconstitution buffer:
  • If the protein is supplied without carrier protein, use sterile 0.1 M acetic acid or sterile distilled water at a minimum concentration of 100 µg/mL.
  • If carrier protein (e.g., BSA) is present, PBS or water may be used, or follow the manufacturer’s datasheet for specific recommendations.
• Dissolve protein: Add the buffer to the vial to achieve your desired stock concentration (typically 0.1–1.0 mg/mL). Gently mix by pipetting or slow vortexing. If the protein does not dissolve immediately, allow it to sit at room temperature for 15–30 minutes with gentle agitation; if flakes remain, mix for up to 2 hours.
• Sterile filtration: If the protein is not supplied sterile, filter the solution through a 0.22 µm sterile filter before use in cell culture to prevent contamination.
• Aliquot and storage: Divide the reconstituted protein into single-use aliquots to avoid repeated freeze-thaw cycles. Store aliquots at −20°C or −80°C for long-term storage, or at 2–8°C for short-term use (up to 1 week).
• Working solution: Dilute the stock solution into your cell culture medium immediately before use. Avoid prolonged exposure to room temperature and repeated freeze-thaw cycles to maintain protein activity.

Additional notes:

• Always consult the product’s Certificate of Analysis (CoA) or datasheet for specific instructions, as formulations may vary.
• If using for bioassays, some protocols recommend adding 0.1% BSA to the buffer to stabilize the protein and prevent adsorption to plasticware.
• For functional assays, typical working concentrations range from 1–100 ng/mL, but optimal dosing should be determined empirically for your cell type and application.

Summary Table: Common Reconstitution Buffers and Concentrations

Always verify the recommended buffer and concentration for your specific recombinant PlGF-1 preparation.