Recombinant Human IL-8/CXCL8 (Mucin Stalk Chimera) offers several compelling advantages for research applications, particularly in studies involving inflammation, cell signaling, and tumor biology.

Enhanced Stability and Bioactivity

The mucin stalk chimera design provides superior stability compared to native IL-8. This fusion protein combines the N-terminal human IL-8 domain (Ala23-Ser99) with the human fractalkine mucin-like stalk domain (Phe103-Gln341), creating a construct that maintains robust biological activity while offering improved handling characteristics. The protein demonstrates potent chemoattractant activity, with an ED50 of 5-25 ng/mL for attracting BaF3 mouse pro-B cells transfected with human CXCR2.

Validated Research Applications

This recombinant protein has been extensively validated for bioactivity assays and is suitable for multiple experimental approaches. The high purity (>95% by SDS-PAGE under reducing conditions) ensures consistent and reproducible results across experiments. The protein is supplied in a lyophilized format from a 0.2 μm filtered solution in PBS with BSA as a carrier protein, facilitating long-term storage and ease of reconstitution.

Functional Versatility

IL-8 plays critical roles in several biological processes relevant to cancer research and inflammation studies. The protein induces VEGF expression, promotes vascular endothelial cell proliferation, enhances angiogenesis, and increases tumor cell invasiveness. In epithelial-mesenchymal transition (EMT) studies, recombinant IL-8 treatment of breast cancer cells results in reduced E-cadherin expression, increased fibronectin expression, and upregulation of mesenchymal markers, making it valuable for investigating tumor progression mechanisms.

Immobilization Compatibility

The C-terminal 6-His tag enables straightforward immobilization on protein G-coated surfaces or anti-His-tag antibody-coated substrates, facilitating dynamic cell-surface interaction studies and high-throughput screening applications. This feature is particularly useful for examining neutrophil responses and integrin-dependent adhesion dynamics.

The combination of enhanced stability, validated bioactivity, high purity, and experimental flexibility makes this chimeric construct an excellent choice for inflammation research, cancer biology, and cell signaling investigations.

You can use recombinant human IL-8 (Mucin Stalk Chimera) as a standard for quantification or calibration in ELISA assays, but only if the ELISA antibodies recognize the chimeric form equivalently to native IL-8 and the standard curve is validated for your assay conditions.

Key considerations:

• Antibody Specificity: Most commercial ELISA kits for human IL-8 are designed to detect the canonical forms of IL-8, typically the 72 or 77 amino acid mature protein. The mucin stalk chimera contains additional sequence elements (from fractalkine) fused to the IL-8 core, which may alter epitope presentation. If your ELISA antibodies are specific for epitopes present in the native IL-8 region (Ala23-Ser99), and these are intact in the chimera, detection should be possible. However, if the antibodies are sensitive to conformational changes or require the native N- or C-terminus, signal may differ.

• Validation Required: Before using the chimera as a standard, you must validate that its dose-response curve in your ELISA matches that of the native IL-8 standard. This involves running parallel standard curves with both proteins and confirming comparable sensitivity, linearity, and recovery.

• Reporting: If you use the chimera, clearly state the protein’s identity and sequence in your methods section, as results may not be directly comparable to assays calibrated with canonical IL-8.

• Formulation: The chimera is typically supplied lyophilized in PBS with BSA, which is compatible with most ELISA protocols. Ensure your diluent matches the matrix used for standard curve generation.

Best Practices:

• Run a side-by-side comparison of the mucin stalk chimera and a canonical IL-8 standard in your ELISA.
• Confirm that the standard curve generated with the chimera is linear and overlaps with the curve from the native protein.
• Document any differences in sensitivity or recovery, and adjust your quantification accordingly.

Summary:
The recombinant human IL-8 (Mucin Stalk Chimera) can be used as an ELISA standard if validated for your specific assay, but you must ensure antibody compatibility and standard curve equivalence before relying on it for quantification.

Applications of Recombinant Human IL-8/CXCL8 (Mucin Stalk Chimera)

Validated Research Applications

The Recombinant Human IL-8/CXCL8 (Mucin Stalk Chimera) has been validated for bioactivity assays as its primary research application. Specifically, the protein's bioactivity is measured through its ability to chemoattract BaF3 mouse pro-B cells transfected with human CXCR2, with an ED50 (effective dose for 50% response) ranging from 5-25 ng/mL.

Mechanistic Research Applications

Beyond basic bioactivity validation, this recombinant protein has been utilized in published research to investigate IL-8's functional roles:

Angiogenic Studies: Recombinant human IL-8 has been employed to demonstrate chemotactic properties on human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells, establishing its proangiogenic effects. The protein has also been used in in vivo models including rat mesenteric window assays, corneal assays, and subcutaneous sponge models to confirm angiogenic activity.

Integrin-Mediated Adhesion Research: The mucin stalk chimera variant has been specifically immobilized on protein G-coated beads for studying the timing and dynamics of integrin activation, particularly examining LFA-1 and Mac-1 signaling responses to immobilized chemokine stimulation.

Receptor Interaction Studies: Research has utilized this protein to investigate IL-8 receptor specificity, particularly distinguishing between CXCR1 and CXCR2-mediated responses in endothelial cell migration and neovascularization.

Technical Specifications Supporting Research Use

The protein is supplied at high purity (>95% by SDS-PAGE) with low endotoxin levels (<1.0 EU per 1 μg), making it suitable for sensitive cell-based assays. It is available in both lyophilized formulations with and without carrier proteins, allowing researchers to select the appropriate form for their specific experimental requirements.

To reconstitute and prepare Recombinant Human IL-8 (Mucin Stalk Chimera) protein for cell culture experiments, follow these general guidelines based on manufacturer protocols and best practices:

Reconstitution

1. Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
2. Reconstitute the protein with sterile PBS (phosphate-buffered saline, pH 7.4). For most products, the recommended concentration is 25 μg/mL (or as specified in the Certificate of Analysis or product datasheet).
   • Example: For a 25 μg vial, add 1 mL of sterile PBS.
   • If the product contains a carrier protein (such as BSA), reconstitute in PBS with at least 0.1% human or bovine serum albumin to stabilize the protein.
3. Gently mix by swirling or tapping the vial; avoid vigorous pipetting or vortexing to prevent protein denaturation.

Preparation for Cell Culture

1. Aliquot the reconstituted protein into small, single-use volumes to minimize freeze-thaw cycles.
2. Store aliquots at -20°C to -80°C for up to 3 months (under sterile conditions).
3. For short-term use, store at 2–8°C for up to 1 month (under sterile conditions).
4. Thaw aliquots on ice or at 4°C before use.
5. Dilute the reconstituted protein in cell culture medium or buffer as needed for your experiment.

Additional Notes

• Avoid repeated freeze-thaw cycles to maintain protein activity.
• Sterile technique is essential to prevent contamination.
• Check the Certificate of Analysis (CoA) or product datasheet for specific instructions, as protocols may vary slightly between suppliers.

By following these steps, you can ensure optimal activity and stability of Recombinant Human IL-8 (Mucin Stalk Chimera) for your cell culture experiments.