Recombinant Human Amphiregulin

Recombinant Human Amphiregulin (AREG) is widely used in research applications due to its critical roles in cell signaling, tissue repair, cancer biology, and immune regulation. Its recombinant form enables precise, reproducible studies of these biological processes.

Key scientific reasons to use recombinant human Amphiregulin include:

• Cell Signaling and Proliferation: Amphiregulin is a member of the epidermal growth factor (EGF) family and binds to the EGF receptor (EGFR), activating downstream pathways such as MAPK and PI3K/AKT. This regulates cell proliferation, survival, migration, and angiogenesis, making it essential for studies on epithelial cell biology, tissue regeneration, and cancer.

• Cancer Research: Amphiregulin is overexpressed in several cancers (breast, colon, pancreas, stomach) and is implicated in tumor growth, metastasis, and therapy resistance. Recombinant AREG is used to model these processes, investigate mechanisms of resistance, and explore targeted therapies.

• Immune Regulation and Tissue Repair: Amphiregulin orchestrates tissue homeostasis by promoting repair after acute or chronic inflammation. It is expressed by immune cells involved in type 2 responses and is critical for restoring tissue integrity following infection or injury. Recombinant AREG is used to study immune tolerance, tissue regeneration, and inflammatory responses.

• Stem Cell and Reproductive Biology: Amphiregulin stimulates growth and differentiation of various cell types, including keratinocytes, mammary epithelial cells, and granulosa cells. It is used in protocols for in vitro maturation (IVM) of oocytes and in vitro fertilization (IVF) to enhance developmental outcomes.

• Functional Assays and Bioactivity Studies: Recombinant AREG is essential for bioassays, ELISA standards, and cell culture experiments to quantify its effects on cell proliferation, differentiation, and signaling.

• Predictive Biomarker Research: Amphiregulin levels can predict treatment resistance in certain cancers, making recombinant AREG valuable for biomarker validation and personalized medicine studies.

Best practices for using recombinant human Amphiregulin:

• Select the appropriate isoform and carrier (e.g., BSA for cell culture) for your application.
• Validate bioactivity in your specific cell type or assay system.
• Use in controlled concentrations to model physiological or pathological conditions.

In summary, recombinant human Amphiregulin is a versatile tool for dissecting mechanisms of cell growth, tissue repair, immune modulation, and cancer progression, supporting a wide range of experimental and translational research applications.

Yes, recombinant human Amphiregulin can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately known. This is a common practice in commercial ELISA kits and in custom assay development.

Supporting details:

• Commercial ELISA kits for Amphiregulin routinely use recombinant human Amphiregulin as the standard to generate calibration curves for quantification in biological samples. For example, the Quantikine ELISA kit uses E. coli-derived recombinant human Amphiregulin as its standard, and validation data show that the standard curve generated with recombinant protein is parallel to curves obtained with natural Amphiregulin, indicating equivalence for quantification purposes.
• Matched antibody pair kits and other ELISA platforms also specify that both natural and recombinant Amphiregulin can be quantified using their systems, further supporting the use of recombinant protein as a standard.
• Key requirements for using recombinant Amphiregulin as a standard:
  • The recombinant protein should be of high purity and free from contaminants that could interfere with antibody binding.
  • The concentration of the recombinant standard must be accurately determined, typically by absorbance at 280 nm or amino acid analysis.
  • The recombinant standard should be reconstituted and stored according to best practices to maintain stability and activity.
  • The standard curve should be prepared in the same matrix as your samples (e.g., buffer, serum, plasma) to minimize matrix effects.

Best practices:

• Prepare a serial dilution of the recombinant Amphiregulin to generate a standard curve covering the expected range of your samples.
• Validate that the standard curve is linear and that sample curves are parallel to the standard curve, which confirms assay accuracy and comparability between recombinant and endogenous Amphiregulin.
• Use duplicate or triplicate wells for standards and samples to ensure precision and reliability.

Limitations and considerations:

• If your recombinant Amphiregulin differs in glycosylation or other post-translational modifications from the native protein in your samples, there may be minor differences in antibody recognition, but most commercial ELISAs are validated to ensure equivalence between recombinant and natural forms.
• Always consult the specific ELISA kit manual or validation data to confirm compatibility with your recombinant standard.

In summary, using recombinant human Amphiregulin as a standard is scientifically valid and widely accepted for ELISA quantification, provided you follow best practices for standard preparation and assay validation.

Recombinant Human Amphiregulin has been validated for a range of applications in published research, primarily in the context of cell signaling, cancer biology, and tissue regeneration.

Key validated applications include:

• Bioassays / Functional Assays: Used to assess biological activity, such as stimulation of cell proliferation (e.g., keratinocytes, mammary epithelial cells, fibroblasts), induction of signaling pathways (EGFR/ERK/NF-κB), and modulation of immune cell function.
• ELISA (Enzyme-Linked Immunosorbent Assay): Used as a standard or antigen to quantify amphiregulin levels in biological samples or to measure antibody responses.
• Western Blot: Used as a positive control or to validate antibody specificity for amphiregulin detection.
• Cell Culture / Stimulation: Added to cell cultures to study effects on cell growth, differentiation, or gene expression, including in cancer cell lines, granulosa cells, and keratinocytes.
• Blocking Assays: Used to investigate the effects of amphiregulin inhibition, often in combination with neutralizing antibodies, to study its role in disease models such as psoriasis and cancer.
• Mass Cytometry (CyTOF) and Spatial Biology: Applied in advanced single-cell and spatial analysis platforms to study amphiregulin’s role in tissue microenvironments.

Representative research applications:

• Cancer Biology: Investigating amphiregulin’s role in tumor growth, metastasis, therapy resistance, and as a biomarker for targeted therapies.
• Psoriasis and Skin Biology: Studying its involvement in epidermal hyperplasia and as a therapeutic target in psoriasis models.
• Reproductive Biology: Exploring its function in granulosa cell signaling, steroidogenesis, and oocyte maturation.
• Immunology: Assessing its effect on regulatory T cell function and immune modulation.
• Tissue Repair and Regeneration: Examining its role in epithelial and mesenchymal cell proliferation during tissue repair, such as after lung injury.

Summary Table of Validated Applications

These applications are supported by both product validation data and peer-reviewed research, demonstrating the versatility of recombinant human amphiregulin in experimental biology.

To reconstitute and prepare Recombinant Human Amphiregulin protein for cell culture experiments, follow these best-practice steps:

• Centrifuge the vial briefly before opening to ensure all lyophilized protein is at the bottom.
• Reconstitution buffer: Use sterile PBS (phosphate-buffered saline) or sterile distilled water. If the protein is supplied carrier-free, it is strongly recommended to add a carrier protein such as 0.1% human or bovine serum albumin (BSA) to enhance stability and prevent adsorption to plastic surfaces.
• Concentration: Reconstitute to a final concentration of 100 μg/mL (0.1 mg/mL) unless your protocol or datasheet specifies otherwise. For some applications, concentrations between 0.1–1.0 mg/mL are also acceptable.
• Dissolving: Add the appropriate volume of buffer, gently swirl or invert the vial, and allow the protein to dissolve for 15–30 minutes at room temperature. Avoid vigorous shaking or vortexing to prevent protein denaturation or foaming.
• Aliquoting: Once fully dissolved, aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade the protein.
• Storage: Store aliquots at –20°C to –80°C for long-term use. For short-term use (up to 1 month), store at 4°C. Avoid multiple freeze-thaw cycles.

Summary protocol:

1. Briefly centrifuge the vial.
2. Add sterile PBS (with 0.1% BSA) to achieve 100 μg/mL.
3. Gently mix and let dissolve at room temperature for 15–30 minutes.
4. Aliquot and store at –20°C to –80°C.

Additional notes:

• If your application is sensitive to animal-derived components, use recombinant or human serum albumin as the carrier.
• For cell culture, always use sterile technique and filter-sterilize the final solution if necessary.
• Consult the specific product datasheet for any unique requirements, as formulations may vary between suppliers.

These steps will ensure Amphiregulin is properly reconstituted and ready for reliable use in cell culture experiments.