Recombinant Human E-Selectin

Recombinant Human E-Selectin (CD62E) is a valuable tool for research applications due to its critical role in mediating cell adhesion and signaling processes, particularly in inflammation, immune responses, and cancer metastasis. Here are several reasons why you should consider using Recombinant Human E-Selectin in your research:

1. Study of Leukocyte-Endothelial Interactions

E-Selectin is a key adhesion molecule expressed on activated endothelial cells. It mediates the initial attachment and rolling of leukocytes (white blood cells) on the blood vessel wall during inflammation. Using recombinant E-Selectin allows you to:

• Model and study leukocyte rolling and adhesion under controlled conditions.
• Investigate the molecular mechanisms of cell recruitment in inflammatory diseases.

2. Investigation of Cancer Metastasis

E-Selectin is involved in the adhesion of circulating tumor cells (CTCs) to the endothelium, a critical step in metastasis. Recombinant E-Selectin enables:

• Analysis of tumor cell adhesion and transendothelial migration.
• Screening for inhibitors or therapeutic agents that block metastatic spread.

3. Binding Assays and Protein-Protein Interactions

Recombinant E-Selectin can be used in binding assays to:

• Characterize interactions with its ligands (e.g., sialyl Lewis X, CD44 glycovariants).
• Study the effects of glycosylation or mutations on binding affinity.

4. Functional Studies in Cell Culture

Immobilized recombinant E-Selectin can induce adhesion of various cell types (e.g., U937 cells, endothelial progenitor cells) in a dose-dependent manner. This is useful for:

• Assessing cell adhesion and migration.
• Evaluating the effects of drugs or genetic modifications on cell behavior.

5. Development of Therapeutic and Diagnostic Tools

E-Selectin is a target for drug delivery and molecular imaging due to its upregulation in inflamed and tumor tissues. Recombinant E-Selectin can be used to:

• Develop and test targeted therapies or imaging agents.
• Validate the specificity and efficacy of E-Selectin inhibitors or ligands.

6. Flow Cytometry and Cell Sorting

Recombinant E-Selectin can be used in flow cytometry to:

• Detect and quantify E-Selectin ligand expression on cell surfaces.
• Isolate specific cell populations based on their adhesion properties.

7. High Purity and Consistency

Recombinant E-Selectin is produced in controlled systems (e.g., CHO cells), ensuring high purity and batch-to-batch consistency, which is essential for reproducible experimental results.

8. Versatility in Applications

Recombinant E-Selectin is suitable for a wide range of applications, including:

• Bioassays
• Binding assays
• Cell culture studies
• Enzyme assays
• Flow cytometry

Summary

Using Recombinant Human E-Selectin in your research allows you to investigate critical biological processes related to inflammation, immune responses, and cancer metastasis. Its versatility, high purity, and well-characterized function make it an essential tool for advancing our understanding of cell adhesion and developing new therapeutic strategies.

Yes, recombinant human E-Selectin can be used as a standard for quantification or calibration in ELISA assays, provided it is highly purified and its concentration is accurately determined. This approach is widely accepted in research applications for quantifying E-Selectin in biological samples such as serum, plasma, and cell culture supernatants.

Essential context and supporting details:

• Recombinant E-Selectin as ELISA Standard: Many commercial ELISA kits for human E-Selectin use recombinant E-Selectin as the calibrator or standard, demonstrating its suitability for generating standard curves and quantifying both recombinant and natural E-Selectin in samples.
• Purity and Quantification: For accurate calibration, the recombinant protein should be highly purified. Its concentration must be precisely measured, typically by spectrophotometry or HPLC, to ensure reliable standard curve generation.
• Parallelism and Specificity: ELISA kits are validated to show that standard curves generated with recombinant E-Selectin are parallel to those obtained with natural E-Selectin, indicating that the assay can accurately quantify both forms.
• Carrier-Free vs. Carrier-Added: Carrier-free recombinant E-Selectin is generally preferred for ELISA calibration to avoid interference from carrier proteins such as BSA, unless the assay specifically requires a carrier for stability.
• Research Use Only: Recombinant E-Selectin standards are intended for research use and not for diagnostic procedures.

Best practices:

• Prepare serial dilutions of the recombinant E-Selectin in the same buffer or diluent used for your samples to minimize matrix effects.
• Validate the standard curve for linearity and parallelism with your sample matrix.
• Store aliquots of the standard at recommended conditions to maintain stability and activity.

Limitations:

• Ensure the recombinant E-Selectin matches the isoform and glycosylation state relevant to your assay, as post-translational modifications may affect antibody recognition in some ELISA formats.
• Always refer to your specific ELISA protocol for compatibility and recommended standard preparation.

In summary, recombinant human E-Selectin is a scientifically accepted standard for ELISA quantification, provided it is properly prepared and validated for your assay system.

Recombinant Human E-Selectin (CD62E) has been validated in published research for a wide range of applications, primarily focused on studying cell adhesion, signaling, and immune responses. The key applications include:

• Binding Assays / Protein-Protein Interaction: Used to study the interaction of E-selectin with its ligands (e.g., PSGL-1, CD44/HCELL) and to measure cell binding under static and flow conditions.
• Bioassays: Employed to assess the functional activity of E-selectin in cell adhesion, migration, and signaling, including studies on leukocyte and cancer cell interactions.
• Flow Cytometry: Utilized to detect and quantify E-selectin binding to cells, such as hematopoietic stem/progenitor cells (HSPCs), endothelial cells, and cancer cells.
• Cell Culture: Applied in studies involving cell adhesion, migration, and differentiation, including the assessment of E-selectin’s role in endothelial and immune cell function.
• Enzyme Assays: Used in some contexts to study E-selectin-mediated signaling pathways.
• Cell Rolling Assays: Employed under physiological flow conditions to mimic in vivo leukocyte rolling on endothelial surfaces.
• Glycan Array Analysis: Used to profile E-selectin ligand specificity and glycan binding.
• Surface Plasmon Resonance (SPR): Applied to measure the kinetics of E-selectin–ligand interactions.
• Western Blot: Used to confirm E-selectin expression and ligand binding in various experimental setups.
• Immunoprecipitation: Employed to isolate and study E-selectin complexes.
• Functional Studies in Disease Models: Used in studies of inflammation, cancer metastasis, thrombosis, and vascular injury, including in vivo and ex vivo models.

These applications are supported by studies using recombinant E-selectin produced in various expression systems (CHO, E. coli, silkworm), and the protein is frequently used as a tool to investigate the molecular mechanisms of cell adhesion and signaling in both normal and pathological conditions.

To reconstitute and prepare Recombinant Human E-Selectin protein for cell culture experiments, follow these general steps, adapting as needed based on your specific product datasheet:

1. Centrifuge the vial briefly (e.g., 10,000 rpm for 1 minute) to ensure all lyophilized protein is at the bottom of the tube before opening.

2. Reconstitution buffer: Most recombinant E-Selectin proteins are reconstituted in sterile phosphate-buffered saline (PBS), pH 7.2–7.4, or sterile distilled water if PBS is not specified. Always check your product datasheet for the recommended buffer.

3. Protein concentration: Reconstitute to a final concentration of 0.1–1.0 mg/mL for general use. For example, add 100–1000 μL of buffer per 100 μg of protein, depending on your desired working concentration.

4. Dissolution: Gently mix by inverting or pipetting up and down. Do not vortex or shake vigorously to avoid foaming and protein denaturation. Allow the vial to sit at room temperature for 10–30 minutes to ensure complete dissolution.

5. Aliquoting: If not using immediately, aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles.

6. Storage:

   • Short-term: Store at 2–8 °C for up to 1 week.
   • Long-term: Store at –20 °C or –70 °C for up to several months.
   • Avoid repeated freeze-thaw cycles to maintain protein integrity.

7. Sterility: If using for cell culture, ensure all solutions and handling are sterile. If the protein is not supplied sterile, filter the reconstituted solution through a 0.2 μm filter before use.

8. Carrier protein (optional): For dilute solutions (<0.1 mg/mL), consider adding a carrier protein such as 0.1% BSA or HSA to stabilize the protein, unless this interferes with your assay.

Example protocol for reconstituting lyophilized recombinant human E-Selectin:

1. Briefly centrifuge the vial to collect the powder.2. Add sterile PBS (pH 7.2–7.4) to achieve a concentration of 0.5 mg/mL.3. Gently mix and let stand at room temperature for 15–30 minutes.4. Aliquot and store at –20 °C or –70 °C.5. Filter sterilize if required for cell culture.

Application note: For cell adhesion assays, E-Selectin is often coated onto tissue culture plates at concentrations such as 2 μg/mL in PBS, incubated at 4 °C overnight, then washed before adding cells.

Always consult the lot-specific datasheet for your recombinant protein, as formulation and recommended reconstitution conditions may vary between suppliers and batches.