Recombinant Mouse P-Selectin

Using Recombinant Mouse P-Selectin in research applications is essential for studying the molecular mechanisms of leukocyte recruitment, inflammation, thrombosis, and vascular biology in murine systems. This protein enables precise, reproducible assays to dissect P-selectin-mediated cell adhesion, signaling, and therapeutic targeting relevant to mouse models.

Key scientific reasons to use recombinant mouse P-selectin include:

• Modeling Leukocyte-Endothelial Interactions: P-selectin is a critical adhesion molecule expressed on activated endothelial cells and platelets, mediating the initial "rolling" of leukocytes on the vascular endothelium—a key step in inflammation and immune cell trafficking. Recombinant protein allows controlled in vitro and in vivo studies of these interactions using mouse cells or tissues, ensuring species-specific relevance.

• Ligand Binding and Inhibition Studies: Recombinant mouse P-selectin is used to characterize binding to its primary ligand, PSGL-1, and to test inhibitors or blocking antibodies that disrupt this interaction. This is crucial for understanding and modulating inflammatory responses in mouse models.

• Functional Assays and Bioassays: The protein is widely used in cell adhesion assays, flow-based rolling assays, and bioassays to quantify leukocyte binding, rolling, and signaling under static or physiological flow conditions. These assays are foundational for dissecting the molecular basis of inflammation, thrombosis, and vascular injury in mice.

• Therapeutic and Mechanistic Studies: Recombinant mouse P-selectin enables the evaluation of anti-inflammatory or anti-thrombotic therapies targeting the P-selectin/PSGL-1 axis in preclinical mouse models. For example, soluble recombinant P-selectin can act as a decoy, competing with endogenous P-selectin and modulating leukocyte recruitment and vascular inflammation.

• Species-Specificity: Mouse and human P-selectin differ in their regulation and function. Using recombinant mouse P-selectin ensures that experimental findings are directly relevant to murine biology and disease models, avoiding cross-species artifacts.

• Protein Engineering and Structural Studies: Recombinant forms (e.g., Fc chimeras, His-tagged proteins) facilitate purification, detection, and immobilization for structural, biophysical, or high-throughput screening applications.

In summary, recombinant mouse P-selectin is a versatile tool for mechanistic, functional, and translational research in mouse models of inflammation, immunity, and vascular disease, providing species-appropriate reagents for robust and reproducible experimentation.

Yes, recombinant Mouse 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 is a common practice in quantitative ELISA protocols for measuring both recombinant and endogenous P-Selectin levels.

Essential context and best practices:

• Standard Curve Preparation: For quantitative ELISA, a standard curve must be generated using a purified protein standard. Recombinant proteins are frequently used for this purpose, as they offer consistency and defined concentration.
• Purity and Quantification: The recombinant Mouse P-Selectin should be highly purified, and its concentration should be precisely measured, ideally by methods such as HPLC or spectrophotometry, to ensure accurate calibration.
• Matrix Considerations: If your samples are complex (e.g., serum, plasma), ensure the diluent used for the standard matches or is compatible with your sample matrix to minimize matrix effects.
• Parallelism: It is important to verify that the standard curve generated with recombinant Mouse P-Selectin is parallel to the response obtained from natural Mouse P-Selectin in your samples. This ensures that the assay quantifies both forms equivalently.
• Lot-to-Lot Consistency: Always use a freshly prepared standard for each assay and follow the reconstitution instructions specific to your recombinant protein lot.
• Validation: If using a recombinant standard not supplied with a commercial ELISA kit, validate its performance in your assay by comparing recovery and linearity with known concentrations of endogenous P-Selectin.

Additional relevant information:

• Many commercial Mouse P-Selectin ELISA kits include recombinant Mouse P-Selectin as the standard for calibration, confirming its suitability for this application.
• Recombinant standards are not recommended for bioassays (functional assays) unless specifically validated for such use, but they are appropriate for ELISA quantification.
• For best results, consult the assay protocol and ensure your recombinant Mouse P-Selectin matches the form (e.g., full-length, extracellular domain) recognized by the antibodies in your ELISA.

Summary: Recombinant Mouse P-Selectin is widely accepted as a standard for ELISA quantification, provided it is properly validated, accurately quantified, and compatible with your assay system.

Recombinant Mouse P-Selectin has been validated for several key applications in published research, primarily in bioassays, in vivo models, cell adhesion studies, and drug delivery systems.

Validated Applications in Published Research:

• Bioassays:
  Used extensively to study leukocyte-endothelial and leukocyte-platelet interactions, especially in the context of inflammation, thrombosis, and vascular injury.
  Examples include:

  • Analysis of neutrophil integrin activation in vivo.
  • Adhesion assays to evaluate T cell homing and recruitment.
  • Investigation of leukocyte rolling and trafficking mechanisms.

• In Vivo Models:
  Applied in mouse models to assess the role of P-selectin in disease processes such as:

  • Acute liver injury and hepatitis, using P-selectin-conjugated liposomes for targeted drug delivery.
  • Restenosis and neointimal formation after arterial injury, particularly in diabetic models.
  • Tumor homing and microenvironment modulation in cancer models.
  • Deep vein thrombosis resolution and leukocyte infiltration.

• Cell Adhesion and Migration Studies:
  Utilized to characterize the adhesion properties of various immune cells (e.g., neutrophils, T cells, microglia) to endothelial cells or platelets, and to study the molecular mechanisms underlying cell recruitment during inflammation and cancer progression.

• Drug Delivery Systems:
  Recombinant P-selectin has been conjugated to nanoparticles or liposomes to enable targeted delivery of therapeutics to P-selectin-expressing tissues, such as inflamed or cancerous sites.

• Protein-Protein Interaction Assays:
  Used to investigate binding interactions with ligands such as PSGL-1 and to screen for inhibitors or modulators of P-selectin function.

Additional Context:

• Sample Types:
  Validated in whole cells, whole tissues, and in vivo mouse models.
• Disease Models:
  Applications span cardiovascular disease, cancer, inflammation, thrombosis, and liver injury.
• Mechanistic Studies:
  Enables mechanistic dissection of P-selectin’s role in immune cell recruitment, vascular biology, and tissue-specific pathology.

Summary Table of Applications

These applications demonstrate the versatility of recombinant mouse P-selectin as a tool for dissecting cellular and molecular mechanisms in immunology, vascular biology, and targeted therapeutics.

To reconstitute and prepare Recombinant Mouse P-Selectin protein for cell culture experiments, dissolve the lyophilized protein in sterile buffer—commonly sterile PBS or distilled water—to a working concentration suitable for your assay, typically between 0.1–1.0 mg/mL. Use aseptic technique throughout to maintain sterility.

Step-by-step protocol:

1. Equilibrate materials:
   Allow the vial of lyophilized protein and your chosen buffer (sterile PBS or distilled water) to reach room temperature before opening.

2. Centrifuge the vial:
   Briefly centrifuge the vial to collect all powder at the bottom, minimizing loss.

3. Add buffer:

   • For most recombinant proteins, add sterile PBS or distilled water.
   • The recommended concentration for reconstitution is typically 100 μg/mL in sterile PBS for mouse P-Selectin.
   • If your experiment requires a different concentration, adjust the volume accordingly (e.g., for 100 μg protein, add 1 mL buffer for 100 μg/mL).

4. Mix gently:
   Gently agitate or swirl the vial to dissolve the protein. Avoid vigorous vortexing, which may denature the protein.

5. Incubate:
   Allow the solution to sit at room temperature for 15–30 minutes. If undissolved flakes remain, continue gentle mixing for up to 2 hours.

6. Aliquot and storage:

   • Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can degrade activity.
   • Store aliquots at −20 °C to −70 °C for long-term storage. For short-term use, 2–8 °C is acceptable for up to one month under sterile conditions.

Additional notes for cell culture:

• If using for cell stimulation, dilute the stock solution in sterile cell culture medium immediately before use.
• Avoid carrier proteins (e.g., BSA) unless required for stability, as they may affect cell responses.
• Filter-sterilize the final working solution if sterility is critical and the buffer/protein is compatible.

Summary Table:

Always consult the specific product datasheet for any unique requirements regarding buffer composition or concentration, as recombinant proteins may vary in formulation and stability.