Recombinant Human Epiregulin

Using Recombinant Human Epiregulin (EREG) in research applications is valuable because it is a biologically active growth factor that modulates cell proliferation, differentiation, and signaling, with roles in tissue regeneration, cancer biology, and stem cell research.

Key scientific reasons to use recombinant human EREG include:

• Cell Proliferation and Differentiation: EREG stimulates the proliferation of various cell types, including fibroblasts, keratinocytes, hepatocytes, vascular smooth muscle cells, and mesenchymal stem cells. It also promotes osteogenic differentiation and inhibits neurogenic trans-differentiation in adipose-derived mesenchymal stem cells, making it useful for tissue engineering and regenerative medicine studies.

• Growth Factor Signaling: EREG is a ligand for the epidermal growth factor receptor (EGFR) and ErbB4, activating downstream pathways such as MEK/ERK and JNK, which are critical for cell growth and survival. Recombinant EREG can be used to dissect these signaling pathways in vitro.

• Cancer Research: EREG is implicated in tumor progression, chemoresistance, and the tumor microenvironment. It acts as a tumor-promoting factor and a potential biomarker for treatment response in oncology research. Targeting EREG or studying its effects can help elucidate mechanisms of cancer cell proliferation, resistance, and stromal interactions.

• Stem Cell Biology: Recombinant EREG enhances the proliferation of dental and mesenchymal stem cells and is involved in maintaining stem cell niches, such as in the intestine and during tissue regeneration. It is used to study stem cell maintenance, differentiation, and tissue modeling.

• Disease Modeling and Functional Assays: EREG is used in bioassays to model developmental niches, fibrosis, and tissue-specific responses, enabling the study of disease mechanisms and therapeutic interventions.

• Reproducibility and Consistency: Recombinant proteins provide a defined, consistent, and animal-free source of EREG, essential for reproducible experimental results and mechanistic studies.

In summary, recombinant human EREG is a versatile tool for investigating cell signaling, proliferation, differentiation, cancer biology, stem cell function, and tissue regeneration due to its well-characterized bioactivity and relevance in multiple physiological and pathological processes.

Recombinant Human Epiregulin can be used as a standard for quantification or calibration in ELISA assays, provided it is compatible with your assay system and matches the native protein’s immunoreactivity. Recombinant standards are commonly used in ELISA kits for quantitative measurement of analytes, including epiregulin.

Key considerations:

• Assay Compatibility: Most commercial human epiregulin ELISA kits are validated to detect both natural and recombinant human epiregulin, and their protocols typically include recombinant protein as the standard for calibration.
• Formulation: Recombinant epiregulin is available in carrier-free and BSA-containing formulations. For ELISA standard use, the BSA-containing formulation is generally recommended due to increased stability, unless BSA interferes with your assay.
• Calibration Curve: Prepare a serial dilution of the recombinant epiregulin standard according to your ELISA kit’s instructions to generate a calibration curve for quantification.
• Validation: Ensure the recombinant protein’s sequence, folding, and post-translational modifications (if relevant) are similar to the endogenous protein detected by your assay antibodies. Most kits specify compatibility with recombinant standards, but if developing a custom assay, validation is required.

Best Practices:

• Use the recombinant standard supplied or recommended by your ELISA kit manufacturer for optimal accuracy.
• If using a recombinant standard from another source, confirm its compatibility with your assay antibodies and detection system.
• Measure standards and samples in duplicate or triplicate to ensure reliability.

Limitations:

• Recombinant proteins expressed in different systems (e.g., E. coli vs. mammalian cells) may differ in glycosylation or folding, potentially affecting antibody recognition.
• Always consult your ELISA kit’s documentation for specific recommendations regarding standard preparation and use.

In summary, recombinant human epiregulin is suitable as a standard for ELISA quantification, but attention to formulation, compatibility, and validation is essential for accurate calibration.

Recombinant Human Epiregulin has been validated primarily for bioactivity assays in published research, with applications focused on its role as a growth factor and signaling molecule in various cellular and tissue models. The following are the main validated applications and experimental contexts:

• Cell Proliferation Assays: Recombinant human epiregulin has been widely used to stimulate proliferation in multiple cell types, including:

  • Fibroblasts (e.g., Balb/3T3 mouse embryonic fibroblasts)
  • Smooth muscle cells and hepatocytes
  • Epidermal keratinocytes and mesangial cells
  • Basal progenitor cells in human and gorilla cortical organoids
  • Dental stem cells from the apical papilla (SCAPs), where it promotes mesenchymal stem cell proliferation

• Bioassays for Growth Factor Activity: Used to assess its ability to activate EGFR and ErbB4 signaling, leading to downstream effects such as cell proliferation, dedifferentiation, or inhibition of tumor cell growth.

• Functional Studies in Organoids and Tissue Models:

  • Human cortical organoids: Epiregulin treatment increases basal progenitor proliferation, and its ablation reduces this effect.
  • Intestinal organoids: Used to study epithelial reprogramming and tissue regeneration.

• Signal Transduction Analysis: Recombinant epiregulin is used to activate and study downstream signaling pathways (e.g., MEK/Erk, JNK) in various cell types, often assessed by Western blotting for phosphorylated proteins.

• Disease Modeling and Therapeutic Research:

  • Cancer research: Used in studies of head and neck squamous cell carcinoma to assess oncogenic signaling and drug sensitivity.
  • Fibrosis and immune signaling: Investigated as a dendritic cell–derived ligand involved in skin and lung fibrosis.
  • Metabolic disease: Studied as an alternative ligand for the leptin receptor in glucose intolerance models.

• SDS-PAGE and Protein Characterization: Validated for use in SDS-PAGE to confirm purity and molecular weight.

• Other Bioactivity-Related Applications:

  • Inhibition studies: Used in combination with blocking antibodies or inhibitors to dissect EGFR-dependent signaling.
  • Cell survival and differentiation: Explored in trophoblast survival and stem cell differentiation contexts.

Summary Table of Validated Applications

Key Points:

• The most common and well-validated application is as a bioactive growth factor in cell-based assays.
• It is frequently used to study cell proliferation, differentiation, and signaling in both normal and disease models.
• Additional uses include protein characterization and as a tool in mechanistic studies of EGFR/ErbB4 signaling.

No evidence was found for routine use in ELISA, Western blot as a detection reagent, or immunoassays; its primary validation is for functional and bioactivity assays.

Reconstitution Protocol

Recombinant Human Epiregulin is supplied as a lyophilized powder and requires proper reconstitution before use in cell culture experiments. The reconstitution process is straightforward but requires attention to specific details to maintain protein activity and stability.

Initial Preparation

Before reconstituting, briefly centrifuge the vial to concentrate the lyophilized powder at the bottom of the tube. This ensures you are working with all the protein material and prevents loss during the reconstitution process. Remove the plastic cap from the vial and gently tap down any loose powder on the sides to consolidate it.

Diluent Selection and Reconstitution Concentration

Reconstitute the lyophilized Epiregulin in sterile, high-purity water (18 MΩ-cm H₂O) or sterile phosphate-buffered saline (PBS). The standard reconstitution concentration is 100 μg/mL, though some protocols recommend concentrations between 0.1 to 1.0 mg/mL depending on your experimental requirements. For formulations containing carrier protein, reconstitute in sterile PBS containing at least 0.1% human or bovine serum albumin.

Reconstitution Procedure

Using a sterile syringe and needle, draw up the calculated volume of diluent needed based on your vial size and desired final concentration. For example, if you have a 750 microgram vial and want a 1 mg/mL solution, draw up 750 microliters of diluent. Insert the needle into the rubber stopper without touching the lyophilized product itself. Dispense the liquid slowly into the vial without forcing it, allowing air to equalize the pressure as needed. Once all liquid is dispensed, gently swirl the vial while avoiding foam formation. Do not vortex the solution, as this can denature the protein.

Storage and Stability

Lyophilized Protein Storage

Store the unopened lyophilized vial at -20 to -70°C for up to 12 months from the date of receipt. Short-term storage at 4°C (up to 6 months) or room temperature (up to 30 days) is permissible if necessary.

Reconstituted Protein Storage

After reconstitution, store working aliquots at -20 to -80°C for extended storage, with a shelf life of approximately 3 months under these conditions. If stored at 2 to 8°C under sterile conditions, the reconstituted protein remains stable for approximately 1 month. Critically, avoid repeated freeze-thaw cycles, as these can compromise protein integrity and biological activity. Use a manual defrost freezer rather than an automatic defrost model to minimize temperature fluctuations.

Biological Activity Considerations

The reconstituted Epiregulin protein is biologically active and will stimulate proliferation in fibroblast cell lines, with an ED₅₀ (effective dose for 50% response) of 0.125-0.75 ng/mL in Balb/3T3 mouse embryonic fibroblasts. The mature secreted form comprises approximately 50 amino residues with a molecular weight of 5.6 kDa. This information is useful for determining appropriate working concentrations for your specific cell culture experiments.