Recombinant Human E-Cadherin

Recombinant Human E-Cadherin is a valuable tool for a wide range of research applications due to its critical role in cell adhesion, tissue homeostasis, and cell signaling. Here are several key reasons why you should consider using Recombinant Human E-Cadherin in your research:

1. Study of Cell Adhesion and Epithelial Integrity

E-Cadherin is a central component of adherens junctions, mediating calcium-dependent homophilic adhesion between epithelial cells. Recombinant E-Cadherin allows you to:

• Investigate the molecular mechanisms of cell-cell adhesion.
• Model epithelial barrier formation and maintenance.
• Study the effects of E-Cadherin clustering and junction assembly in vitro.

2. Investigation of Contact Inhibition and Cell Proliferation

E-Cadherin homophilic ligation is known to inhibit cell growth and promote contact inhibition, a process essential for tissue homeostasis and suppression of uncontrolled proliferation. Using recombinant E-Cadherin, you can:

• Trigger and study density-dependent growth inhibition.
• Examine the signaling pathways downstream of E-Cadherin engagement.
• Compare the effects of E-Cadherin ligation in primary cells and cancer cell lines.

3. Research on Cancer and Metastasis

Loss of E-Cadherin is a hallmark of epithelial-to-mesenchymal transition (EMT) and cancer metastasis. Recombinant E-Cadherin enables:

• Functional studies on how E-Cadherin expression or loss affects tumor cell behavior.
• Investigation of E-Cadherin’s role in suppressing migration and invasion.
• Development of assays to screen for compounds or antibodies that modulate E-Cadherin function.

4. Stem Cell and Regenerative Medicine Applications

E-Cadherin is crucial for the self-renewal and pluripotency of human pluripotent stem cells (hPSCs). Recombinant E-Cadherin can be used to:

• Support the culture and differentiation of hPSCs in defined, xeno-free conditions.
• Study the role of E-Cadherin in stem cell niche formation and maintenance.
• Develop substrates for stem cell expansion and organoid formation.

5. Angiogenesis and Tumor Microenvironment Studies

Soluble E-Cadherin (sE-cad) has been shown to promote angiogenesis and influence tumor microenvironment dynamics. Recombinant E-Cadherin allows you to:

• Investigate the pro-angiogenic effects of sE-cad on endothelial cells.
• Study the role of E-Cadherin in tumor-stroma interactions.
• Develop assays to block or modulate E-Cadherin-mediated angiogenesis.

6. Bioassays and Functional Studies

Recombinant E-Cadherin is suitable for a variety of bioassays, including:

• Cell adhesion assays (e.g., with MCF-7 cells).
• Spheroidogenesis and 3D culture models.
• ELISA and Western blot controls.
• Functional assessment of cell signaling and junction formation.

7. Defined and Reproducible Experimental Conditions

Using recombinant E-Cadherin provides a defined, consistent, and reproducible source of the protein, which is essential for:

• Standardizing experiments across different labs.
• Reducing variability associated with cell-derived or tissue-extracted proteins.
• Enabling high-throughput screening and drug discovery.

In summary, Recombinant Human E-Cadherin is a versatile and essential reagent for studying cell adhesion, tissue homeostasis, cancer biology, stem cell biology, and angiogenesis. Its use can enhance the rigor, reproducibility, and translational relevance of your research.

You can use recombinant human E-Cadherin as a standard for quantification or calibration in ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately known, and it is compatible with the antibodies used in your assay.

Supporting details:

• Many commercial ELISA kits for human E-Cadherin use recombinant human E-Cadherin as the standard for generating calibration curves, enabling quantification of both recombinant and native E-Cadherin in samples.
• The standard curve in these assays is typically constructed using serial dilutions of recombinant E-Cadherin, and sample concentrations are interpolated from this curve.
• ELISA kits specify that they can detect and quantify both native and recombinant E-Cadherin in biological samples, indicating that recombinant protein is suitable as a standard.
• It is important to ensure that the recombinant E-Cadherin you use matches the form recognized by the capture and detection antibodies in your assay (e.g., full-length extracellular domain, correct folding, and post-translational modifications if relevant).
• The accuracy of quantification depends on the purity and concentration determination of your recombinant standard. Use a well-characterized, preferably carrier-free recombinant protein, and verify its concentration using a reliable method (e.g., absorbance at 280 nm, BCA assay).

Best practices:

• Prepare a serial dilution of the recombinant E-Cadherin in the same buffer or matrix as your samples to minimize matrix effects.
• Confirm that your ELISA antibodies recognize the recombinant form you are using (some antibodies may be conformation- or epitope-specific).
• Validate the linearity and recovery of your standard curve in the context of your specific assay and sample types.

Limitations:

• If your recombinant E-Cadherin differs significantly in structure or post-translational modifications from the endogenous protein, this could affect antibody recognition and quantification accuracy.
• Always consult the ELISA kit documentation or antibody datasheets to confirm compatibility with recombinant standards.

In summary, recombinant human E-Cadherin is widely used and accepted as a standard for ELISA quantification, provided it is appropriately validated for your specific assay system.

Recombinant Human E-Cadherin has been validated for a range of applications in published research, primarily in studies of cell adhesion, cell signaling, cancer biology, and stem cell biology.

Key validated applications include:

• Cell Adhesion Assays: Recombinant E-Cadherin is widely used to support and quantify cell-cell adhesion, particularly in epithelial cell lines such as MCF-7 breast cancer cells. Immobilized E-Cadherin on culture substrates has been shown to mediate calcium-dependent adhesion, mimicking physiological adherens junctions.

• Bioassays/Functional Assays: Numerous studies have used recombinant E-Cadherin in functional bioassays to investigate:

  • The role of E-Cadherin in epithelial-to-mesenchymal transition (EMT) and cancer metastasis.
  • Modulation of collective cell migration and tissue organization, including the use of E-Cadherin-coated surfaces to study collective epithelial dynamics.
  • Interactions with other proteins, such as catenins and integrins, and their impact on cell signaling and cytoskeletal organization.
  • The effect of E-Cadherin on the derivation and maintenance of pluripotent stem cells, including xeno-free derivation of human embryonic stem cells.

• ELISA (Enzyme-Linked Immunosorbent Assay): Recombinant E-Cadherin is used as a standard or capture protein in ELISA to quantify soluble E-Cadherin or to study protein-protein interactions.

• Western Blot Control: Cell lysates or recombinant protein are used as positive controls in Western blotting to validate E-Cadherin antibodies and to assess protein expression in various cell types.

• Cell Culture Substrate: Surfaces functionalized with recombinant E-Cadherin have been used to study how cadherin-mediated adhesion influences cell behavior, including migration, proliferation, and tissue morphogenesis.

• Cancer Research Models: Recombinant E-Cadherin is employed to investigate its tumor suppressor functions, its loss in cancer progression, and its potential as a therapeutic target in various cancers (e.g., breast, prostate, bladder, and gastric cancers).

• Stem Cell Research: It has been used to facilitate the derivation and maintenance of pluripotent stem cells, supporting cell adhesion in defined, xeno-free conditions.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and product validation data, demonstrating the versatility of recombinant human E-Cadherin in both basic and translational research contexts.

Reconstitution Protocol

Recombinant Human E-Cadherin proteins are typically supplied in lyophilized form and require proper reconstitution before use in cell culture experiments. The reconstitution process is critical for maintaining protein functionality and stability.

Initial Preparation

Before opening the vial, centrifuge it briefly to collect any lyophilized material at the bottom. This ensures you recover all the protein during reconstitution.

Reconstitution Conditions

The reconstitution concentration and diluent depend on the specific protein formulation:

• Standard E-Cadherin protein: Reconstitute to a concentration of 0.1–0.5 mg/mL in sterile distilled water
• E-Cadherin Fc Chimera protein: Reconstitute at 100 μg/mL in sterile DPBS (Dulbecco's phosphate-buffered saline) containing Ca²⁺ and Mg²⁺
• Alternative formulations: Some preparations may require reconstitution in PBS or other buffers as specified in the certificate of analysis

Handling Precautions

When reconstituting the protein, avoid vortexing or vigorous pipetting, as these actions can denature the protein and reduce its biological activity. Instead, gently mix the solution by pipetting slowly or allowing it to dissolve gradually.

Storage and Stability

Short-term Storage

After reconstitution, store the protein solution at 2–8°C for up to one week. At this temperature, the protein remains stable for immediate use in experiments.

Long-term Storage

For extended storage, maintain the reconstituted protein at −20°C, where it remains stable for approximately 3 months. For storage periods exceeding 3 months, use a −70°C freezer.

Freeze-Thaw Considerations

Avoid repeated freeze-thaw cycles, as these can compromise protein integrity and reduce adhesion activity. If you anticipate multiple uses, consider aliquoting the reconstituted protein into smaller portions to minimize freeze-thaw exposure.

Stabilizer Addition

For long-term storage, consider adding carrier proteins or stabilizers such as 0.1% bovine serum albumin (BSA) or 5% human serum albumin (HSA) to the reconstituted solution.

Application-Specific Considerations

For cell adhesion assays, the optimal concentration depends on your specific cell type and experimental objectives. As a reference, E-Cadherin Fc Chimera coated at 1.5 µg/mL supports >60% cell adhesion of certain cell lines after 90 minutes at 37°C. Titration experiments are recommended to determine the ideal concentration for your particular cell culture system.