Recombinant Mouse CD62E

Using Recombinant Mouse CD62E (E-selectin) in research applications is valuable for studying leukocyte-endothelial interactions, inflammation, immune cell trafficking, and disease models involving vascular adhesion. CD62E is a key adhesion molecule expressed on activated endothelial cells, mediating the initial attachment and rolling of leukocytes on the vascular wall during inflammation.

Key scientific applications and benefits include:

• Modeling Leukocyte Adhesion and Trafficking: Recombinant CD62E enables in vitro assays to study how leukocytes bind and roll on endothelium, a critical step in immune surveillance and inflammation. This is essential for dissecting the molecular mechanisms of cell adhesion and migration.

• Bioassays and Binding Studies: The recombinant protein can be immobilized to support adhesion assays, such as measuring the binding of leukocyte cell lines (e.g., U937 cells) or primary immune cells, allowing quantification and mechanistic analysis of cell adhesion under controlled conditions.

• Cancer and Metastasis Research: CD62E-mediated adhesion is implicated in tumor cell extravasation and metastasis. Recombinant CD62E can be used to model and block these interactions, aiding in the development of anti-metastatic therapies and understanding tumor-vascular interactions.

• Hematopoietic Stem Cell (HSC) Niche Studies: E-selectin in the bone marrow niche regulates HSC activation, proliferation, and survival. Recombinant CD62E is useful for investigating how niche signals influence stem cell fate and for testing therapeutic strategies that target these interactions.

• Inflammation and Vascular Biology: Recombinant CD62E is used to study the molecular basis of inflammatory diseases, vascular injury, and endothelial activation, as well as to screen for inhibitors of leukocyte recruitment.

• Flow Cytometry and Cell Sorting: CD62E can be used as a ligand in flow cytometry to identify and sort cells expressing E-selectin ligands, such as cutaneous lymphocyte-associated antigen (CLA) on T cells.

• Therapeutic Target Validation: By using recombinant CD62E in blocking or competition assays, researchers can validate potential therapeutic antibodies or small molecules that inhibit E-selectin-mediated adhesion, relevant for anti-inflammatory or anti-metastatic drug development.

In summary, Recombinant Mouse CD62E is a versatile tool for dissecting the cellular and molecular mechanisms of leukocyte-endothelial interactions, inflammation, stem cell biology, and cancer metastasis, and for validating therapeutic interventions targeting vascular adhesion pathways.

You can use recombinant Mouse CD62E (E-selectin) as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately known, and it is compatible with your assay system.

Key considerations:

• Purity and Quantification: The recombinant protein should be highly purified and its concentration precisely determined, as impurities or inaccurate quantification can compromise the accuracy of your standard curve.
• Formulation: If your recombinant CD62E is supplied with stabilizers (e.g., BSA or other carriers), ensure these do not interfere with your assay or differ from the matrix of your samples.
• Standard Curve Preparation: Prepare a fresh standard curve for each assay using serial dilutions of the recombinant protein in the same buffer or diluent as your samples to ensure matrix compatibility and minimize variability.
• Assay Validation: Confirm that the recombinant standard produces a linear, parallel response in your ELISA compared to native CD62E in biological samples. This ensures that the recombinant protein is recognized equivalently by the assay antibodies.
• Documentation: Many commercial ELISA kits for mouse CD62E use recombinant protein as the standard, and their protocols specify that a new standard curve must be generated for each assay.

Best Practices:

• Use the same diluent for standards and samples.
• Validate the standard curve range to cover expected sample concentrations.
• Store and handle recombinant protein according to manufacturer or best laboratory practices to maintain stability and activity.

Limitations:

• If your recombinant CD62E differs in glycosylation or other post-translational modifications from native protein, there may be minor differences in antibody recognition, though this is generally not a major issue for most sandwich ELISAs.
• Always check that your ELISA antibodies are reactive with the recombinant form you are using.

In summary, recombinant Mouse CD62E is suitable as a standard for ELISA quantification, provided you follow standard curve preparation and assay validation protocols.

Recombinant Mouse CD62E (E-selectin) has been validated for multiple applications in published research, primarily including bioassays, binding assays, flow cytometry, ELISA, and in vivo studies.

Key validated applications:

• Bioassays: Used to study cell adhesion, leukocyte rolling, and trafficking, especially in the context of immune cell interactions with endothelial cells. Examples include assays measuring T cell homing, stem cell adhesion, and chemokine-guided migration.

• Binding Assays: Employed to quantify and characterize the interaction between E-selectin and its ligands, such as glycoforms of CD43 or engineered peptides on stem cells.

• Flow Cytometry: Utilized for detecting E-selectin binding to specific cell populations, such as CLA(+) CD4(+) T cells, and for analyzing cell surface expression and ligand interactions.

• ELISA: Used as a standard or capture reagent for quantifying E-selectin or its ligands in biological samples.

• In Vivo Studies: Applied in mouse models to investigate the role of E-selectin in processes like leukocyte recruitment, vascular niche-mediated chemoresistance, and stem cell homing.

• Cell Adhesion Assays: Recombinant E-selectin coated on surfaces induces adhesion of cell lines (e.g., U937 cells), measured by colorimetric or other quantitative assays.

• Western Blot: Used for detection and quantification of E-selectin in cell lysates or tissue samples.

• Preclinical Therapeutic Evaluations: Serves as a reagent in studies of cancer metastasis, cardiovascular disease, and tissue regeneration, particularly for evaluating cell migration and vascular interactions.

Additional research areas include:

• Cancer biology: Investigating tumor microenvironment and metastasis.
• Cardiovascular research: Studying endothelial activation and atherosclerosis.
• Regenerative medicine: Engineering stem cells for targeted delivery to inflamed tissues via E-selectin-mediated homing.

These applications are supported by published studies using recombinant mouse CD62E in both in vitro and in vivo experimental systems, confirming its utility in immunology, cell biology, and translational research.

To reconstitute and prepare Recombinant Mouse CD62E (E-Selectin) protein for cell culture experiments, follow these steps:

• Reconstitution: Add sterile, deionized water to the lyophilized protein to achieve a final concentration of 1 mg/mL. Gently mix by pipetting up and down or by slow vortexing. Avoid vigorous agitation to prevent protein denaturation.

• Buffer Considerations: The protein is typically lyophilized from a PBS solution. If your application is sensitive to buffer composition, you may further dilute or exchange into your desired buffer (e.g., cell culture medium or PBS) after initial reconstitution.

• Aliquoting and Storage: After reconstitution, aliquot the protein to avoid repeated freeze-thaw cycles, which can reduce activity. Store aliquots at -20°C to -80°C for long-term storage, or at 2–8°C for short-term use (up to 1 month). For extended storage, adding a carrier protein such as 0.1% BSA is recommended to stabilize the protein, especially at lower concentrations.

• Working Concentration: For cell-based assays, typical coating concentrations range from 2 µg/mL (for plate-based adhesion assays) up to 10 µg/mL (for binding assays). The optimal concentration should be determined empirically for your specific application.

• Sterility: Ensure all solutions and materials are sterile to prevent contamination in cell culture experiments.

Summary protocol:

1. Add sterile, deionized water to the lyophilized protein to reach 1 mg/mL.
2. Mix gently until fully dissolved.
3. Aliquot and store at -20°C or colder; avoid repeated freeze-thaw cycles.
4. Dilute to the desired working concentration in sterile buffer or medium immediately before use.

Additional notes:

• Do not reconstitute to concentrations below 100 µg/mL unless necessary, as lower concentrations may reduce stability.
• If using for plate coating, incubate plates with the protein solution (e.g., 2 µg/mL, 100 µL/well) at room temperature or 4°C, then wash before adding cells.
• Always consult the specific product datasheet for any unique instructions or recommendations.

These guidelines ensure optimal protein stability and activity for cell culture experiments involving recombinant mouse CD62E.