Recombinant Human Epigen

Recombinant Human Epigen is used in research applications primarily because it is a potent, specific mitogen for epithelial cells and fibroblasts, making it valuable for studying cell proliferation, tissue repair, and EGFR signaling pathways. As a member of the EGF superfamily, Epigen offers unique biological properties distinct from other EGFR ligands, including persistent signaling and potential roles in cancer biology and wound healing.

Key reasons to use Recombinant Human Epigen in research:

• Mitogenic Activity: Epigen robustly stimulates the proliferation of epithelial cells and fibroblasts, which is essential for assays investigating cell growth, tissue regeneration, and wound healing mechanisms.
• EGFR Pathway Studies: Epigen binds to the epidermal growth factor receptor (EGFR/ErbB1), activating downstream signaling cascades such as MAPK, which are central to cell proliferation, differentiation, and survival.
• Cancer Research: Epigen is overexpressed in certain cancers (e.g., breast, prostate, bladder), and its expression correlates with tumor invasiveness and therapeutic response, making it a relevant model for studying tumor biology and drug sensitivity.
• Unique Biological Features: Unlike other EGFR ligands, Epigen exhibits low-affinity receptor binding but prolonged membrane persistence, resulting in sustained signaling and sub-maximal receptor activation, which may more closely mimic physiological conditions.
• Wound Healing and Regenerative Medicine: Due to its ability to promote epithelial cell growth, Epigen is a candidate for exploring novel wound-healing therapies and tissue engineering approaches.
• High Purity and Consistency: Recombinant production ensures high purity, batch-to-batch consistency, and the absence of animal-derived contaminants, which are critical for reproducible and reliable experimental results.

Additional applications include:

• Bioassays: Used as a positive control or stimulant in cell proliferation assays.
• Drug Screening: Useful for identifying and characterizing EGFR pathway inhibitors or modulators.
• Structural and Functional Studies: Facilitates investigation of ligand-receptor interactions and downstream signaling mechanisms.

In summary, Recombinant Human Epigen is a versatile tool for research in cell biology, oncology, regenerative medicine, and pharmacology due to its potent, specific, and well-characterized biological effects on epithelial and fibroblast cells.

Yes, recombinant human Epigen can be used as a standard for quantification or calibration in ELISA assays, provided it is of sufficient purity and its concentration is accurately determined. This is a common practice in quantitative ELISA protocols for proteins where purified recombinant forms are available.

Key considerations and best practices:

• Purity and Identity: The recombinant Epigen should be highly purified and well-characterized, ideally matching the native protein’s sequence and post-translational modifications relevant to your assay.
• Concentration Determination: Accurately measure the concentration of your recombinant standard, preferably using orthogonal methods such as absorbance at 280 nm, BCA assay, or HPLC.
• Standard Curve Preparation: Prepare a serial dilution of the recombinant Epigen in the same buffer or diluent used for your samples. Typical standard curve ranges for Epigen ELISA kits are from low ng/mL to 100 ng/mL, but this should be optimized for your assay’s expected sample concentrations.
• Validation: Confirm that your ELISA antibodies recognize the recombinant Epigen equivalently to the endogenous protein. Most commercial Epigen ELISA kits use recombinant Epigen as the standard and have validated their antibodies for this purpose.
• Matrix Effects: If your samples are in complex matrices (e.g., serum, plasma, tissue lysate), consider spiking experiments to assess recovery and parallelism between the recombinant standard and endogenous Epigen.
• Documentation: Follow the manufacturer’s instructions for reconstitution and dilution of the recombinant standard, as protocols may be lot-specific and affect quantification accuracy.

Limitations:

• Quantification accuracy may be affected by differences between recombinant and native protein forms, especially if post-translational modifications or folding differ.
• Large dilution steps can introduce error; meticulous pipetting and validation are required.
• For research use only: Recombinant standards and ELISA kits are typically not validated for diagnostic use.

Summary Table: Recombinant Epigen as ELISA Standard

In conclusion, recombinant human Epigen is suitable as a standard for ELISA quantification if these best practices are followed and the assay is properly validated for your specific application.

Recombinant Human Epigen (EPGN) has been validated for several applications in published research, primarily focusing on its biological activity and use in functional assays. The main applications include:

• Bioactivity Assays: The protein is validated for bioactivity, specifically for its ability to stimulate the proliferation of murine Balb/3T3 cells in a dose-dependent manner. This is a standard assay for assessing the mitogenic activity of growth factors.
• Functional Assays: Epigen is used in functional assays to study its effects on cell proliferation and differentiation, particularly in epithelial cells and fibroblasts.
• SDS-PAGE: The protein is validated for use in SDS-PAGE for purity assessment.
• HPLC: High-performance liquid chromatography is used for purity and quality control.
• Western Blotting: Epigen is used in Western blotting to detect and quantify the protein in various samples.
• Mass Cytometry (CyTOF): The protein has been used in mass cytometry for detailed cellular analysis.
• Spatial Biology: Epigen is applied in spatial biology studies to understand its distribution and function in tissues.
• Vaccine Development: There are applications in vaccine development, although specific details are not provided in the sources.

These applications highlight the versatility of Recombinant Human Epigen in both basic research and more specialized areas such as cancer biology and tissue engineering.

To reconstitute and prepare Recombinant Human Epigen protein for cell culture experiments, dissolve the lyophilized protein in phosphate-buffered saline (PBS) containing at least 0.1% human or bovine serum albumin (BSA) to a final concentration of 100 μg/mL.

Essential steps and best practices:

• Preparation:

  • Before opening, briefly centrifuge the vial to ensure all powder is at the bottom.
  • Use sterile technique throughout to prevent contamination.

• Reconstitution:

  • Add sterile PBS with 0.1% BSA directly to the vial to reach the desired concentration (typically 100 μg/mL).
  • Gently mix by swirling or inverting; avoid vigorous shaking or vortexing to prevent protein denaturation and foaming.
  • Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation.

• Carrier Protein:

  • The addition of BSA (0.1%) is recommended to stabilize the protein and prevent adsorption to tube surfaces, especially for low concentrations or long-term storage.

• Aliquoting and Storage:

  • Once fully dissolved, aliquot the solution into working volumes to avoid repeated freeze-thaw cycles.
  • Store aliquots at −20°C or colder for long-term stability; short-term storage (up to one week) can be at 2–4°C.
  • For extended storage, adding glycerol (5–50%) can further stabilize the protein.

• Working Solution for Cell Culture:

  • Dilute the stock solution in cell culture medium immediately before use.
  • Typical working concentrations for bioassays range from 150–300 ng/mL, based on dose-dependent stimulation of cell proliferation.

Additional notes:

• If the protein is supplied carrier-free, always add BSA or another carrier protein during reconstitution to minimize loss and maintain activity.
• Avoid concentrations below 100 μg/mL during initial reconstitution to prevent precipitation or loss of activity.
• Confirm biological activity using a proliferation assay (e.g., murine Balb/3T3 cells) if required for your experiment.

Summary Table: Reconstitution Protocol

This protocol ensures optimal solubility, stability, and biological activity of recombinant human Epigen protein for cell culture applications.