Recombinant Human ErbB3

Using Recombinant Human ErbB3 in research applications enables precise investigation of ErbB3’s biological functions, signaling mechanisms, and its role in disease models, particularly in cancer, cardiovascular, and developmental biology.

Key reasons to use recombinant ErbB3 include:

• Modeling Ligand-Receptor Interactions: Recombinant ErbB3 allows controlled studies of its binding with ligands such as heregulins (neuregulins), enabling dissection of downstream signaling pathways, especially those involving PI3K/Akt and HER2/ErbB2 heterodimerization.
• Cancer Research: ErbB3 is a critical node in tumorigenesis, drug resistance, and metastasis in several cancers, including breast, lung, and skin cancers. Recombinant ErbB3 can be used to:
  • Study receptor activation and downstream signaling.
  • Screen for inhibitors or antibodies targeting ErbB3.
  • Model the effects of ErbB3 overexpression or mutation in cell-based assays.
• Therapeutic Target Validation: ErbB3 is a promising therapeutic target, with monoclonal antibodies and inhibitors under development. Recombinant protein is essential for in vitro binding, inhibition, and neutralization assays.
• Functional Assays: Recombinant ErbB3 can inhibit the biological activity of its ligand (e.g., NRG1-β1) in cell-based assays, providing a tool to study ligand-dependent signaling and to validate antagonists.
• Biomarker Studies: ErbB3 is upregulated in diseases such as pulmonary hypertension and various cancers, making it a useful biomarker for disease progression and therapeutic response.
• Protein-Protein Interaction Studies: The cytoplasmic domain of ErbB3 contains multiple binding motifs for SH2 and SH3 domain-containing proteins, making recombinant ErbB3 valuable for mapping signaling complexes and interaction networks.

Additional considerations:

• Recombinant ErbB3 is typically used in its extracellular domain form for ligand-binding and neutralization studies, or as full-length protein for signaling and functional assays.
• It is not suitable for therapeutic or diagnostic use in humans or animals, but strictly for research applications.

In summary, recombinant human ErbB3 is a versatile tool for elucidating the molecular mechanisms of ErbB3 function, validating drug targets, and modeling disease processes where ErbB3 plays a pivotal role.

Yes, recombinant human ErbB3 (HER3) can be used as a standard for quantification or calibration in ELISA assays, provided that the recombinant protein is:

• Biologically relevant and structurally similar to the native protein detected by your ELISA kit (e.g., properly folded and, if necessary, glycosylated).
• Compatible with the antibody pair used in your ELISA (i.e., the capture and detection antibodies must recognize the recombinant form).
• Supplied in a known, pure, and quantifiable form (ideally with a certificate of analysis or concentration verification).

Key Considerations

1. Kit Compatibility: Most commercial ELISA kits (such as those from Abcam, R&D Systems, Thermo Fisher, etc.) are designed to detect both natural and recombinant human ErbB3. For example, the Invitrogen (Thermo Fisher) Human ErbB3 ELISA Kit explicitly states that it recognizes both natural and recombinant forms.

2. Standard Curve: To generate a standard curve, serial dilutions of the recombinant ErbB3 should be prepared in the same matrix as your samples (e.g., buffer, serum, or plasma) to minimize matrix effects.

3. Validation: If you are not using the standard provided by the kit manufacturer, it is important to validate that your recombinant ErbB3 generates a dose-response curve with similar sensitivity and linearity as the kit’s native standard.

4. Glycosylation and Isoforms: If your assay is sensitive to post-translational modifications (such as glycosylation), ensure that your recombinant protein matches the expected form (e.g., glycosylated vs. non-glycosylated).

Practical Steps

• Use recombinant ErbB3 to prepare a dilution series (e.g., 0, 10, 30, 100, 300, 1000 pg/mL).
• Run the dilution series alongside your samples.
• Plot the signal (OD) vs. concentration to generate a standard curve.
• Use the curve to interpolate concentrations in your samples.

References

• Many ELISA kit manuals (e.g., Thermo Fisher, Abcam, R&D Systems) state that their assays recognize both natural and recombinant ErbB3.
• If in doubt, consult the product datasheet or contact the manufacturer for confirmation.

In summary, recombinant human ErbB3 is suitable as a standard for ELISA quantification, as long as it is compatible with your assay system and properly validated.

Recombinant Human ErbB3 has been validated for several key applications in published research:

Functional and Binding Assays

ELISA applications represent a primary validated use for recombinant ErbB3 proteins. The recombinant protein has been specifically validated for measuring its ability to inhibit the biological activity of Neuregulin-1-beta 1 (NRG1-β1) on MCF-7 human breast cancer cells, with reported ED50 values in the range of 0.3-3 µg/mL. Additionally, functional ELISA assays have been established to measure binding interactions between recombinant ErbB3 and human NRG1-β1.

Cell-Based Functional Studies

Recombinant ErbB3 has been employed in cell culture and tissue culture applications as a ligand-binding protein to study receptor-ligand interactions. The protein serves as a tool for investigating ErbB3 heterodimerization with other ErbB family members, particularly ErbB2, which is critical for understanding cancer cell signaling and therapeutic resistance mechanisms.

Structural and Biochemical Characterization

SDS-PAGE analysis has been validated as an application for characterizing recombinant ErbB3 preparations, with purity standards exceeding 90% commonly achieved. This enables quality assessment and protein characterization studies.

Therapeutic Development Research

Recombinant ErbB3 has been utilized in drug sensitivity assays to evaluate the efficacy of therapeutic agents targeting ErbB3 and related pathways. These studies have demonstrated the actionability of ErbB3 variants in response to various ErbB-targeting therapeutics, supporting the development of novel treatment strategies for ErbB3-driven cancers.

To reconstitute and prepare Recombinant Human ErbB3 protein for cell culture experiments, dissolve the lyophilized protein in sterile PBS or sterile water to the recommended concentration, typically between 0.1–0.5 mg/mL depending on the specific product and application. Use sterile technique throughout to maintain protein integrity and prevent contamination.

Step-by-step protocol:

• Centrifuge the vial briefly at 4°C before opening to ensure all lyophilized material is collected at the bottom.
• Add sterile buffer: For most ErbB3 preparations, reconstitute in sterile PBS (pH 7.2–7.4) or sterile deionized water. Common recommended concentrations are:
  • 500 μg/mL in PBS
  • 0.415 mg/mL in sterile water
  • 0.5–1.0 mg/mL in sterile deionized water
• Gently mix: Pipette up and down to dissolve. Do not vortex, as this may denature the protein.
• Aliquot: Divide into small volumes to avoid repeated freeze-thaw cycles, which can degrade the protein.
• Storage: Store aliquots at −80°C for long-term use. For frequent use, store at 2–8°C and use within 30 days. Avoid repeated freeze-thaw cycles.

Preparation for cell culture:

• Dilute the stock solution to the desired working concentration using sterile cell culture medium or PBS immediately before use. Typical working concentrations for functional assays range from 10–500 ng/mL, but optimal dosing should be determined empirically for your cell type and assay.
• Filter sterilize if necessary, using a 0.2 μm filter, especially if the protein will be added directly to cell cultures.
• Add to cells: Supplement the culture medium with the recombinant ErbB3 protein as required by your experimental design.

Additional notes:

• Always consult the specific product datasheet for recommended reconstitution buffer and concentration, as formulations may vary.
• If the protein contains stabilizers (e.g., trehalose, mannitol), these are generally compatible with cell culture but verify for your application.
• Confirm protein activity and absence of endotoxin if using in sensitive cell-based assays; endotoxin levels should be <1 EU/μg for most applications.

Summary Table:

This protocol ensures optimal solubility, stability, and bioactivity of recombinant ErbB3 for cell culture experiments.