Recombinant Human P-Selectin

Recombinant human P-selectin offers several compelling advantages for research applications, particularly in studies involving cell adhesion, inflammation, and vascular biology.

Key Research Applications

Cell Adhesion and Leukocyte Interactions

Recombinant P-selectin is essential for investigating leukocyte-endothelial interactions. P-selectin mediates the initial "rolling" of circulating leukocytes on endothelial cells through its interaction with P-selectin glycoprotein ligand-1 (PSGL-1). This makes it invaluable for studying the early stages of leukocyte recruitment during inflammatory responses. The protein can be immobilized on surfaces to create physiologically relevant adhesion assays, with studies demonstrating that immobilized P-selectin induces greater than 50% adhesion of cell lines such as U937 cells.

Inflammatory Pathway Studies

The protein is particularly useful for examining P-selectin's role in inflammation-mediated pathways. Research has demonstrated that P-selectin upregulates tissue factor in monocytes and leads to leukocyte accumulation at sites of vascular injury, contributing to thrombosis and inflammatory responses. By using recombinant P-selectin, you can isolate and study these specific molecular interactions without the complexity of whole-cell systems.

Therapeutic Development

Recombinant P-selectin serves as a critical tool for developing therapeutic interventions. Studies using recombinant P-selectin glycoprotein ligand-Fc (rPSGL-Ig) have demonstrated significant reductions in neointimal hyperplasia and improved vessel patency in disease models. This makes the protein valuable for screening compounds and biologics designed to modulate P-selectin-mediated pathways.

Experimental Versatility

Recombinant P-selectin is compatible with multiple experimental formats. It can be used as a positive control in Western blotting, ELISA, and immunoprecipitation assays. The protein is available in different formats—including Fc-chimera constructs and carrier-free preparations—allowing you to select the appropriate form for your specific application, whether for cell culture, binding assays, or surface plasmon resonance studies.

Storage and Handling Considerations

For optimal results, store the protein at 4°C for short-term use (1-2 weeks) or at -20°C for extended storage. Avoid multiple freeze-thaw cycles to maintain biological activity and protein integrity throughout your experiments.

Yes, recombinant human P-Selectin can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. This approach is widely accepted in quantitative ELISA protocols for soluble proteins such as P-Selectin.

Essential context and supporting details:

• Standard Curve Preparation: ELISA quantification relies on a standard curve generated using known concentrations of the target protein. Recombinant human P-Selectin is commonly used for this purpose, as it provides a consistent and well-characterized standard.
• Purity and Quantification: The recombinant protein should be highly purified, and its concentration should be verified using an orthogonal method (e.g., HPLC, UV absorbance at 280 nm, or BCA assay) to ensure accuracy in calibration.
• Assay Compatibility: Commercial ELISA kits for human P-Selectin routinely use recombinant human P-Selectin as the standard, and validation data show that curves generated with recombinant protein are parallel to those obtained with natural P-Selectin, supporting its suitability for quantification.
• Sample Types: This standardization is applicable for quantifying P-Selectin in plasma, serum, cell culture supernatants, and other biological fluids.
• Best Practices:
  • Prepare serial dilutions of the recombinant standard in the same matrix as your samples (e.g., sample diluent or buffer) to minimize matrix effects.
  • Include replicates and ensure the standard curve covers the expected concentration range of your samples.
  • Confirm that the recombinant standard is soluble and stable under assay conditions.

Additional relevant information:

• Validation: If using a custom recombinant P-Selectin, it is advisable to validate its performance in your specific ELISA system by comparing standard curves with those from a commercial kit or natural protein, if available.
• Reporting: When publishing or reporting results, specify that recombinant human P-Selectin was used as the calibration standard and provide details on its source, purity, and quantification method.

In summary, recombinant human P-Selectin is scientifically appropriate and routinely used as a standard for ELISA quantification, as long as its purity and concentration are well characterized and validated for your assay system.

Recombinant human P-selectin has been validated for numerous applications across diverse research and therapeutic domains:

Cell Adhesion and Leukocyte Interactions

Recombinant P-selectin constructs have been extensively validated for studying cell adhesion mechanisms. The protein effectively induces adhesion in cell-based assays, with Fc chimera formulations demonstrating greater than 50% adhesion on U937 cells in standardized binding assays. These applications are fundamental for investigating leukocyte recruitment and the initial tethering and rolling of leukocytes during inflammatory responses mediated through P-selectin glycoprotein ligand-1 (PSGL-1) interactions.

Protein-Protein Interaction Studies

Recombinant P-selectin serves as a validated tool for binding assays and protein-protein interaction studies. Surface plasmon resonance (SPR) analysis has been employed to determine binding affinity (KD) and kinetic parameters using recombinant P-selectin ligands, enabling precise characterization of molecular interactions. These applications are critical for developing targeted therapeutics and understanding selectin-mediated signaling pathways.

Immunological Applications

The protein functions effectively as a positive control in Western blotting, ELISA, immunoprecipitation, and other immunological experiments. Recombinant P-selectin has been utilized as a capture antigen in standard ELISA protocols for validating binding characteristics of fusion proteins and therapeutic constructs.

Vascular and Inflammatory Disease Models

Recombinant P-selectin glycoprotein ligand Fc (rPSGL-Ig) has demonstrated significant therapeutic potential in diabetes-associated vascular injury models. Administration of this construct reduced P-selectin expression by over 50% and decreased neointimal hyperplasia in balloon-injured carotid arteries of diabetic rats, suggesting applications in restenosis prevention. Similarly, P-selectin-targeted complement inhibitor constructs have been validated in hindlimb ischemia/reperfusion injury models, showing reduced myeloid cell infiltration and protection against tissue damage.

Targeted Drug Delivery

Recombinant P-selectin has been validated as a targeting ligand for nanotherapeutic platforms. Nanoparticles engineered with P-selectin-binding moieties have demonstrated selective dose-dependent binding to recombinant P-selectin while showing minimal cross-reactivity with other selectins or non-specific proteins. These platforms have been validated for targeted delivery of chemotherapeutics and kinase inhibitors to P-selectin-expressing tumors, including melanoma and breast cancer models, resulting in superior antitumor efficacy with reduced off-target toxicity.

Immunological Assays

Recombinant P-selectin serves as a standard for sELISA and other quantitative immunoassays, with His-tagged variants expressed in mammalian systems achieving purity levels exceeding 80%.

To reconstitute and prepare Recombinant Human P-Selectin protein for cell culture experiments, dissolve the lyophilized protein in sterile buffer (commonly sterile PBS or deionized water) at the concentration recommended in the product documentation, typically ranging from 100 μg/mL to 1 mg/mL. Mix gently and avoid vigorous agitation to prevent protein denaturation or foaming.

Step-by-step protocol:

• Equilibrate: Allow the vial and reconstitution buffer to reach room temperature before starting.
• Collect lyophilisate: Briefly centrifuge or tap the vial to ensure all powder is at the bottom.
• Add buffer: Add the precise volume of sterile buffer (PBS or deionized water) indicated on the product datasheet to achieve the desired concentration (e.g., 100 μg/mL or 1 mg/mL).
• Mix gently: Let the vial stand at room temperature for 10–30 minutes, gently swirling or inverting to dissolve. Do not vortex or shake vigorously.
• Aliquot: If not using immediately, aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
• Storage: Store aliquots at −20°C to −80°C for long-term use. For short-term use (up to one week), store at 2–8°C.
• Carrier protein (optional): For enhanced stability, especially at low concentrations, dilute with a carrier protein solution such as 0.1% BSA or 10% FBS, unless your experiment requires serum-free conditions.
• Glycerol (optional): For long-term storage, adding 5–50% glycerol can help maintain protein stability.

Preparation for cell culture:

• Dilution: Before adding to cell culture, dilute the reconstituted P-Selectin to the working concentration in your culture medium. If using serum-containing medium, direct dilution is acceptable. For serum-free or in vivo applications, avoid animal-derived carriers and consider using trehalose for stabilization.
• Sterility: Ensure all buffers and solutions are sterile to prevent contamination.
• Avoid freeze-thaw: Use fresh aliquots for each experiment to maintain protein integrity.

Additional notes:

• Always consult the specific Certificate of Analysis or product datasheet for exact reconstitution instructions, as formulations may vary between preparations.
• If the protein is intended for functional assays (e.g., cell adhesion), confirm activity post-reconstitution using appropriate controls.

This protocol ensures optimal solubility, stability, and biological activity of recombinant P-Selectin for cell culture applications.