Recombinant Human PAI-1 produced in Sf1 insect cells is advantageous for research applications requiring a functionally active, glycosylated form of PAI-1, especially for studies on fibrinolysis, extracellular matrix remodeling, and disease models involving angiogenesis, tumor invasion, or metabolic disorders.

Key scientific reasons to use this reagent include:

• Functional Activity and Glycosylation: Insect cell expression systems (such as Sf1, derived from Spodoptera frugiperda) enable the production of recombinant human PAI-1 with post-translational modifications, including glycosylation, that are essential for its biological activity and stability. This is critical for accurately modeling human physiological processes, as bacterial systems cannot provide these modifications.

• High Purity and Reproducibility: Recombinant PAI-1 from insect cells is typically purified to >95% homogeneity and demonstrates consistent inhibitory activity against target proteases (e.g., uPA, tPA), ensuring reliable results in enzyme assays, bioassays, and cell-based studies.

• Scalability and Cost-Effectiveness: Insect cell cultures are easily scalable, do not require CO₂ supplementation, and can be maintained in serum-free media, making them cost-effective for large-scale protein production.

• Safety and Versatility: Baculovirus vectors used in insect cells are non-replicative in humans, reducing biosafety concerns. The system is versatile for producing recombinant proteins for biochemical, immunological, and therapeutic research.

• Relevant Applications: Recombinant PAI-1 is widely used in:

  • Enzyme inhibition assays to study fibrinolysis and protease regulation.
  • Cell signaling and migration studies, particularly in cancer, fibrosis, and cardiovascular research.
  • Western blotting, immunocytochemistry, and multiplex immunoassays for quantifying PAI-1 in biological samples.
  • Investigating the role of PAI-1 in extracellular matrix remodeling, apoptosis, and metabolic disorders.

• Rapid Turnaround: Insect cell systems allow for quick production cycles, facilitating timely experimental workflows.

In summary, using recombinant human PAI-1 from Sf1 insect cells provides a biologically relevant, active, and glycosylated protein suitable for a wide range of research applications, particularly those requiring human-like post-translational modifications and reliable functional assays.

Yes, Recombinant Human PAI-1 produced in Sf1 insect cells can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for your specific assay format and matches the native protein in terms of immunoreactivity and structure.

Essential context and supporting details:

• Recombinant PAI-1 proteins are commonly used as standards in quantitative ELISA kits for human PAI-1 (SERPINE1). These standards are typically full-length, purified proteins expressed in various systems, including insect cells (Sf1, Sf21), and are suitable for generating standard curves in sandwich ELISA formats.
• The key requirement is that the recombinant protein must be recognized by the capture and detection antibodies used in your ELISA. Most commercial ELISA kits for PAI-1 use recombinant human PAI-1 as their calibration standard, and protocols specify serial dilutions to generate a standard curve for quantification.
• Validation: Before using your recombinant PAI-1 (Sf1 insect cells) as a standard, confirm:
  • The protein is full-length and properly folded (matching the native sequence, e.g., Met1-Pro402).
  • It is free of significant contaminants and has a defined concentration (preferably determined by absorbance at 280 nm or amino acid analysis).
  • It is immunoreactive with the antibodies in your ELISA (test by running a standard curve and comparing to a known standard if possible).
• Assay compatibility: Some ELISA kits specify the use of recombinant PAI-1 standards expressed in mammalian or insect cells, and published protocols demonstrate successful quantification using these proteins. The molecular mass (∼43–44 kDa) and tag (e.g., His-tag) should be noted, as tags may affect antibody binding in rare cases.
• Calibration procedure: Prepare serial dilutions of the recombinant PAI-1 in the assay buffer recommended by your ELISA protocol, covering the expected concentration range (e.g., 0.625–1500 nM or 1.56–100 ng/mL). Use these dilutions to generate a standard curve for quantification of unknown samples.

Additional relevant information:

• If your ELISA is designed for native PAI-1, confirm that the recombinant standard does not differ in glycosylation or conformation in a way that affects antibody recognition. Insect cell-expressed proteins may have different post-translational modifications compared to mammalian cell-expressed proteins, but most commercial ELISA antibodies target linear or conserved epitopes.
• For regulatory or publication purposes, document the source, purity, and validation data for your recombinant standard.
• If using a custom or in-house ELISA, run parallel curves with both your recombinant standard and a commercial standard to ensure equivalency.

In summary, recombinant human PAI-1 from Sf1 insect cells is suitable as an ELISA standard if it is validated for your assay and recognized by the antibodies used. Always confirm compatibility and document your validation process.

Applications of Recombinant Human PAI-1 from Insect Cell Expression

Recombinant human PAI-1 produced in insect cells has been validated for several important research applications across multiple biological domains.

Cell Senescence and Aging Research

PAI-1 serves as both a validated marker and functional mediator of cellular senescence. Research has demonstrated that overexpressed PAI-1 is sufficient to induce replicative fibroblast senescence independently of p53, and this role extends to other cell types including keratinocytes and vascular cells. The protein has been particularly valuable in studying senescence-associated secretory phenotype (SASP) development, with studies showing that PAI-1 inhibition or deletion blocks TGF-β–induced senescence and SASP development in alveolar type II cells.

Cell Migration and Signaling Studies

PAI-1 functions as an integrator of cell signaling and migration pathways. The protein interacts with multiple molecular targets including vitronectin (VN), urokinase plasminogen activator (uPA), uPA receptor (uPAR), and lipoprotein receptor-related protein 1 (LRP1), enabling manipulation of cellular motility programs at multiple mechanistic levels. These interactions make recombinant PAI-1 valuable for investigating the attachment-detachment-reattachment cycle that governs cell migration.

Fibrinolysis and Hemostasis Research

PAI-1 is the principal inhibitor of tissue plasminogen activator (tPA) and plays a central role in the fibrinolytic system. Recombinant PAI-1 has been validated for fibrinolysis studies and plasminogen activation research, making it essential for investigating coagulation and fibrinolytic balance.

Pathophysiological Model Systems

The protein has been applied in research examining pathological conditions including fibrosis, inflammation, hypertension, and metabolic disorders. Studies have demonstrated PAI-1's involvement in bleomycin- and doxorubicin-induced cellular senescence both in vitro and in vivo, with PAI-1 deletion in mice suppressing bleomycin-induced senescence and attenuating lung fibrosis.

Receptor Binding and Protein Interaction Studies

Recombinant PAI-1 specifically binds vitronectin and can be used for affinity purification and receptor binding studies, enabling investigation of protein-protein interactions critical to cell adhesion and signaling pathways.

The insect cell expression system is particularly advantageous for these applications because it enables complex post-translational modifications, including glycosylation, which are essential for maintaining the functional activity of human PAI-1.

To reconstitute and prepare Recombinant Human PAI-1 (sf1 Insect Cells) protein for cell culture experiments, follow these steps:

• Reconstitution:
  Dissolve the lyophilized protein at 500 μg/mL in sterile 50 mM sodium acetate and 100 mM NaCl, pH 5.5.
  Gently mix the solution to ensure complete dissolution. Avoid vigorous vortexing to prevent protein denaturation.

• Handling and Storage:

  • Warm the lyophilized vial to room temperature before opening to minimize condensation.
  • After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles, which can reduce protein activity.
  • Store aliquots at −20 °C or below in a manual defrost freezer.
  • For extended storage, the addition of 0.1% BSA can help stabilize the protein.

• Sterility:
  Use only sterile reagents and equipment throughout the process. The protein should be reconstituted in a sterile environment to maintain suitability for cell culture applications.

• Dilution for Cell Culture:
  After reconstitution, dilute the stock solution to the desired working concentration using sterile cell culture medium or buffer compatible with your experimental design. Ensure the final buffer conditions are compatible with your cells (e.g., physiological pH and osmolarity).

• Endotoxin Consideration:
  The protein is typically supplied with low endotoxin levels (<1.0 EU/μg), making it suitable for cell culture. If your application is highly sensitive to endotoxin, consider additional endotoxin removal steps.

Summary Table: Reconstitution Protocol

Additional Notes:

• Always consult the specific product datasheet for any lot-specific recommendations.
• If you require a different buffer for your cell culture system, perform a buffer exchange (e.g., by dialysis or desalting columns) after initial reconstitution.
• Confirm protein activity after reconstitution if your experiment is activity-dependent.

This protocol ensures optimal solubility, stability, and bioactivity of recombinant PAI-1 for cell culture experiments.