Recombinant Rat E-Selectin

Using Recombinant Rat E-Selectin in research applications is essential for studying the molecular mechanisms of leukocyte adhesion, inflammation, and vascular biology, as well as for modeling disease processes and testing therapeutic interventions in rat systems.

Key reasons to use recombinant rat E-selectin include:

• Modeling Leukocyte-Endothelial Interactions: E-selectin (CD62E) is an inducible adhesion molecule expressed on activated endothelial cells and is critical for the initial recruitment and rolling of leukocytes on the vascular endothelium during inflammation. Recombinant rat E-selectin allows you to reconstitute and study these interactions in vitro, using rat-specific systems.

• Species-Specific Research: Using the rat protein ensures compatibility with rat cells and ligands, which is crucial for translational studies, preclinical models, and for avoiding cross-species artifacts that may arise with human or mouse proteins.

• Functional Assays: Recombinant rat E-selectin is validated for supporting the adhesion of leukocyte cell lines (e.g., U937 cells) in cell adhesion assays, enabling quantitative and mechanistic studies of cell binding, rolling, and signaling under static or flow conditions.

• Inflammation and Disease Models: E-selectin is upregulated in various inflammatory diseases (e.g., psoriasis, arthritis, transplant rejection) and cardiovascular conditions, making it a valuable tool for investigating pathogenesis, biomarker discovery, and therapeutic targeting in rat models.

• Cancer and Metastasis Research: E-selectin mediates interactions between endothelial cells and circulating tumor cells, influencing metastasis. Recombinant rat E-selectin can be used to study these processes and to screen for inhibitors or therapeutic antibodies in rat-based cancer models.

• High Purity and Activity: Recombinant preparations are typically highly purified (>90–97%), endotoxin-controlled, and biologically active, ensuring reproducibility and reliability in experimental assays.

• Versatile Applications: Applications include cell adhesion assays, flow cytometry, ELISA standards, ligand binding studies, and as a control or competitor in functional experiments.

In summary, recombinant rat E-selectin is a critical reagent for dissecting the molecular and cellular basis of inflammation, immune cell trafficking, and vascular pathology in rat models, and for developing and validating new therapeutic strategies targeting selectin-mediated processes.

Yes, recombinant rat E-Selectin can be used as a standard for quantification or calibration in ELISA assays, provided it is sufficiently purified and its concentration is accurately determined. Recombinant proteins are commonly used as standards in ELISA assays to generate calibration curves for quantifying target analytes in biological samples.

Key considerations for using recombinant rat E-Selectin as an ELISA standard:

• Purity and Quantification: The recombinant protein should be highly purified. Its concentration must be accurately measured, typically by absorbance at 280 nm, BCA assay, or HPLC. Impurities or inaccurate quantification can lead to errors in your standard curve.
• Standard Curve Preparation: Prepare serial dilutions of the recombinant E-Selectin to cover the expected range of concentrations in your samples. Most ELISA standard curves range from low picogram to nanogram levels, depending on assay sensitivity.
• Validation: Confirm that the recombinant E-Selectin behaves similarly to native E-Selectin in your assay system. Some ELISA kits are validated to recognize both recombinant and natural forms of rat E-Selectin. Recovery and linearity should be assessed to ensure accurate quantification.
• Compatibility: Ensure the recombinant standard is compatible with the antibodies used in your ELISA. Most commercial rat E-Selectin ELISA kits specify that their antibodies recognize both recombinant and natural forms.
• Documentation: Follow best practices for reconstitution, storage, and handling as recommended for recombinant protein standards.

Limitations and best practices:

• If using a recombinant standard outside a validated kit, you must calibrate it against a mass-calibrated reference standard for highest accuracy.
• Lot-to-lot variability and differences in glycosylation or folding between recombinant and native proteins can affect assay results. Always validate the standard in your specific assay context.
• For publication-quality quantification, document the source, purity, and quantification method of your recombinant standard.

Summary Table: Recombinant E-Selectin as ELISA Standard

In conclusion: Recombinant rat E-Selectin is suitable as a standard for ELISA quantification if properly validated and quantified. Always verify compatibility and performance within your specific assay system.

Recombinant Rat E-Selectin has been validated for several key applications in published research, primarily in studies of cell adhesion, inflammation, angiogenesis, and as a standard in immunoassays.

Validated Applications:

• Cell Adhesion Assays:
  Recombinant rat E-selectin is widely used to study the adhesion of leukocytes (such as U937 human histiocytic lymphoma cells) to endothelial surfaces. Immobilized E-selectin supports the adhesion of these cells in vitro, modeling the physiological process of leukocyte rolling and arrest on activated endothelium. This application is fundamental for dissecting the molecular mechanisms of leukocyte recruitment during inflammation.

• Functional Binding Studies:
  Recombinant E-selectin proteins are used in binding assays (including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays) to characterize interactions with ligands such as HCELL and PSGL-1. These studies help elucidate the structural requirements for E-selectin–ligand binding and the impact of domain architecture on function.

• ELISA and Immunoassays:
  Recombinant rat E-selectin serves as a standard or capture antigen in ELISA kits for quantifying E-selectin levels in serum, plasma, or cell culture supernatants. These assays are used to monitor endothelial activation and inflammation in various disease models.

• Western Blotting:
  Recombinant E-selectin is used as a positive control in Western blot analyses to validate antibody specificity and to quantify E-selectin expression in cell and tissue lysates.

• Angiogenesis and Vascular Biology Research:
  E-selectin has been used in gene therapy and in vitro models to study its role in angiogenesis, vascular remodeling, and tissue perfusion, particularly in ischemic disease models. Recombinant E-selectin is instrumental in dissecting its effects on endothelial cell behavior and neovascularization.

• Inflammation and Immunology Models:
  Recombinant E-selectin is used to investigate its role in leukocyte recruitment, endothelial activation, and inflammatory signaling pathways, including rapid NLRP3 inflammasome activation in neutrophils.

Additional Context:

• Disease Models:
  E-selectin is implicated in inflammatory skin lesions, arthritis, transplant rejection, and cardiovascular disease, and recombinant forms are used to model these conditions in vitro and in vivo.

• Cancer Research:
  E-selectin is studied for its role in cancer cell metastasis and as a potential therapeutic target, with recombinant protein used in functional assays and drug development studies.

Summary Table:

These applications are supported by published research and product validation data, making recombinant rat E-selectin a versatile tool in vascular biology, immunology, and translational research.

To reconstitute and prepare Recombinant Rat E-Selectin protein for cell culture experiments, dissolve the lyophilized protein in sterile, distilled water or buffer to the desired concentration, typically starting at 1 mg/mL, and then dilute to working concentrations (e.g., 2 μg/mL for cell adhesion assays).

Essential protocol steps:

• Reconstitution:

  • Use sterile, distilled water or sterile PBS (pH 7.4) for initial reconstitution unless the product manual specifies a different buffer.
  • Add the appropriate volume to achieve the recommended stock concentration (commonly 1 mg/mL).
  • Gently mix by pipetting or slow inversion; avoid vigorous shaking or vortexing to prevent protein denaturation or foaming.
  • Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation.

• Aliquoting and Storage:

  • Once fully dissolved, aliquot the solution to avoid repeated freeze-thaw cycles.
  • Store aliquots at < –20°C for long-term use (up to 3 months) or at 4–8°C for short-term use (2–7 days).
  • Avoid storing at room temperature for extended periods.

• Preparation for Cell Culture:

  • Dilute the reconstituted stock to the desired working concentration using sterile PBS or cell culture medium. For adhesion assays, a typical coating concentration is 2 μg/mL in 100 μL/well.
  • If coating plates, incubate the diluted protein solution in wells at 37°C for 1 hour, then wash to remove unbound protein.
  • Ensure endotoxin levels are low (< 1.0 EU/μg) to minimize cell activation artifacts.

• General Best Practices:

  • Always consult the specific product manual for buffer recommendations and reconstitution instructions.
  • Use aseptic technique throughout to prevent contamination.
  • If the protein is formulated with stabilizers (e.g., trehalose, mannitol, Tween 80), these do not need to be removed for most cell culture applications.

Summary Table: Recombinant Rat E-Selectin Reconstitution

If your experiment requires a different concentration or buffer, adjust accordingly and always verify protein solubility and activity in your specific assay conditions.