Recombinant Mouse Eotaxin-2 (aa 27-119)

Recombinant Mouse Eotaxin-2 (aa 27-119) is a highly valuable tool for research applications focused on type 2 immunity, allergic inflammation, and eosinophil biology. Here are the key reasons to use this specific recombinant protein:

1. High Biological Activity

• The aa 27-119 variant of Mouse Eotaxin-2 (CCL24) is the most biologically active form. Studies show that the aa 24-119 variant is about 150-fold less active, making the aa 27-119 version the preferred choice for robust chemotactic responses in experimental settings.

2. Selective Chemotaxis for Eosinophils and Other CCR3+ Cells

• Eotaxin-2 selectively chemoattracts cells expressing the CCR3 receptor, including eosinophils, basophils, Th2 T cells, mast cells, and certain dendritic cell subsets. This makes it ideal for studying the recruitment and activation of these cell types in models of allergic inflammation, asthma, and other type 2 immune responses.

3. Well-Validated for Functional Assays

• Recombinant Mouse Eotaxin-2 (aa 27-119) has been validated for bioactivity, including chemotaxis assays using cells such as BaF3 mouse pro-B cells transfected with mouse CCR3. This ensures reliable and reproducible results in your experiments.

4. Low Endotoxin Levels

• Commercially available recombinant Eotaxin-2 (aa 27-119) is typically produced with very low endotoxin levels (<0.01 EU/µg), minimizing the risk of confounding effects from bacterial contaminants in sensitive immunological assays.

5. Relevance to In Vivo Models

• Eotaxin-2 is a key mediator in murine models of allergic airway inflammation. Its expression is induced by allergen challenge and IL-4, and it plays a critical role in eosinophil recruitment and the amplification of type 2 immune responses. Using this protein allows you to mimic or modulate these physiological processes in your research.

6. Consistency and Reproducibility

• Recombinant proteins provide a consistent and defined source of Eotaxin-2, unlike tissue-derived or conditioned media sources, which can vary in concentration and purity. This is essential for controlled experiments and comparative studies.

7. Broad Applicability

• Eotaxin-2 is not only relevant for eosinophil studies but also for investigating the broader roles of chemokines in immune cell trafficking, inflammation, and immune regulation.

In summary, Recombinant Mouse Eotaxin-2 (aa 27-119) is the optimal choice for research applications requiring high biological activity, specificity for CCR3+ cells, and reliable, reproducible results in the study of type 2 immunity and allergic inflammation.

Yes, you can use recombinant Mouse Eotaxin-2 (aa 27-119) as a standard for quantification or calibration in ELISA assays, provided it matches the sequence and form recognized by your assay antibodies.

Supporting details:

• Recombinant Eotaxin-2 (aa 27-119) is commonly used as a standard in commercial ELISA kits for mouse CCL24/Eotaxin-2 quantification. Multiple ELISA kits specify the use of recombinant mouse CCL24/Eotaxin-2, expressed in E. coli and corresponding to amino acids 27-119, as their standard protein.
• The standard curve in these kits is generated using this recombinant protein, with typical ranges from 3.28–1000 pg/mL, and the protein is usually supplied lyophilized and reconstituted in assay buffer.
• Guidelines for ELISA standards recommend using a purified recombinant protein that matches the analyte detected by the assay antibodies. The recombinant protein should be reconstituted and diluted according to the protocol to generate a reliable standard curve.
• The sequence (aa 27-119) is the mature form of mouse Eotaxin-2, which is the relevant region for antibody recognition in most ELISA formats.

Best practices:

• Confirm that your ELISA antibodies (capture and detection) are validated to recognize the recombinant form (aa 27-119) and that there are no sequence mismatches or post-translational modifications required for recognition in your specific assay.
• Prepare the standard curve using serial dilutions of the recombinant protein in the same buffer/matrix as your samples to minimize matrix effects.
• If your ELISA kit or protocol specifies a different isoform or sequence (e.g., aa 24-119), verify cross-reactivity or equivalence before use.

Summary Table: Use of Recombinant Mouse Eotaxin-2 (aa 27-119) as ELISA Standard

Conclusion:
If your ELISA is designed for mouse Eotaxin-2 and validated for the mature protein (aa 27-119), this recombinant protein is suitable as a standard for quantification or calibration. Always follow the specific instructions of your assay protocol for optimal results.

Recombinant Mouse Eotaxin-2 (aa 27-119) has been validated for several key applications in published research, primarily focused on its bioactivity and use in cell-based assays.

Validated Applications:

• Chemoattractant Assays:
  The protein is widely used to measure chemotactic activity, especially for mouse BaF3 pro-B cells transfected with mouse CCR3. The Val27-Val119 isoform is notably potent, being 150-fold more active than the Ile24-Val119 isoform in these assays. This application is central to studies of eosinophil and T lymphocyte migration, as Eotaxin-2 is a strong chemoattractant for these cell types.

• Suppression of Myeloid Cell Proliferation:
  Eotaxin-2 has been shown to suppress the proliferation of multipotential hematopoietic progenitor cells, a function not shared by other eotaxins.

• ELISA Standard:
  Recombinant Mouse Eotaxin-2 (aa 27-119) is used as a standard in ELISA assays for the quantitative measurement of Eotaxin-2 in biological samples. This is essential for studies investigating cytokine profiles in mouse models of inflammation, allergy, and immune response.

• Flow Cytometry:
  The protein is used in flow cytometry protocols to enumerate cells expressing CCR3 or CXCR5, typically via indirect staining methods for signal enhancement.

• Cell Analysis and Functional Studies:
  It is employed in cell analysis methods to study chemokine-receptor interactions, cell migration, and signaling pathways relevant to immunology and allergy research.

Additional Context:

• Research Use Only:
  The recombinant protein is strictly for research use and not validated for diagnostic or therapeutic applications.

• Disease Models:
  Eotaxin-2 is relevant in mouse models of allergic asthma, vernal keratoconjunctivitis, and other conditions involving eosinophil recruitment and activation.

• Protein Characterization:
  The recombinant protein is characterized by high purity (>97% by SDS-PAGE), low endotoxin levels (<0.01 EU/μg), and a predicted molecular mass of 10.3 kDa.

Summary Table of Validated Applications

These applications are supported by published research and product validation data, making Recombinant Mouse Eotaxin-2 (aa 27-119) a standard tool for immunological and cell migration studies in mice.

To reconstitute and prepare Recombinant Mouse Eotaxin-2 (aa 27-119) for cell culture experiments, dissolve the lyophilized protein in sterile PBS to a concentration of 100 μg/mL under aseptic conditions.

Detailed protocol and best practices:

• Reconstitution:

  • Briefly centrifuge the vial to collect the lyophilized powder at the bottom.
  • Add sterile PBS (phosphate-buffered saline) to achieve a final concentration of 100 μg/mL.
  • Gently swirl or invert the vial to mix; avoid vigorous vortexing to prevent protein denaturation.
  • If a different working concentration is required, further dilute the reconstituted stock with sterile PBS or cell culture medium as appropriate for your assay.

• Storage after reconstitution:

  • Store the reconstituted protein at 2–8 °C for up to 1 month under sterile conditions.
  • For longer-term storage (up to 3 months), aliquot and freeze at –20 °C to –70 °C.
  • Avoid repeated freeze-thaw cycles, as these can reduce protein activity.

• General handling tips:

  • Use a manual defrost freezer for storage at subzero temperatures.
  • If the protein is supplied with carrier protein (e.g., BSA), this can help stabilize the protein during storage and handling.
  • Always handle the protein aseptically to prevent contamination, especially if it will be used in cell culture.

• Application in cell culture:

  • Before adding to cells, dilute the reconstituted protein to the desired working concentration using sterile PBS or cell culture medium.
  • The effective concentration for functional assays (e.g., chemotaxis) is typically in the range of 1–5 ng/mL, but this should be optimized for your specific experimental setup.

• Quality and activity:

  • The protein is typically >97% pure by SDS-PAGE and has low endotoxin levels (<0.01 EU/μg), making it suitable for cell-based assays.
  • Biological activity is confirmed by its ability to chemoattract BaF3 mouse pro-B cells transfected with mouse CCR3, with an ED50 of 1–5 ng/mL.

If your protocol or application requires a different buffer or additive (such as carrier protein or reducing agents), ensure compatibility with your downstream assays and cell type. Always consult the specific product datasheet for any lot-specific recommendations.