Recombinant Human GCP-2

Recombinant Human GCP-2 (CXCL6) is a multifunctional chemokine widely used in research for its potent chemotactic, angiogenic, antibacterial, and chondrogenic activities. Its recombinant form ensures high purity, batch-to-batch consistency, and suitability for controlled experimental applications.

Key scientific reasons to use recombinant human GCP-2 in research:

• Neutrophil Chemotaxis and Activation: GCP-2 is a strong chemoattractant for neutrophils, promoting their migration and degranulation via CXCR1 and CXCR2 receptor activation. This makes it valuable for studying immune cell recruitment, inflammation, and host defense mechanisms.

• Antibacterial Activity: GCP-2 exhibits robust antibacterial properties against both Gram-positive and Gram-negative bacteria, with activity up to 90-fold higher than related chemokines (CXCL5, CXCL7). It disrupts bacterial membranes, making it useful for investigating innate immunity and antimicrobial responses.

• Angiogenesis: GCP-2 promotes new blood vessel formation, supporting studies in tissue regeneration, wound healing, and tumor biology.

• Cartilage and Chondrogenesis Research: GCP-2 has anabolic effects on cartilage, enhancing extracellular matrix formation and collagen type II synthesis in human cartilage organoids and in vivo models. It can block chondrocyte hypertrophy and reduce cartilage loss, making it relevant for osteoarthritis and regenerative medicine studies.

• Disease Models: Recombinant GCP-2 and its engineered variants (e.g., GCP-2-T) have been used to modify disease progression and symptoms in osteoarthritis models, demonstrating both anti-inflammatory and tissue-protective effects.

• Quantitative and Functional Assays: Recombinant GCP-2 is essential for ELISA standards, chemotaxis bioassays, and receptor activation studies, enabling precise quantification and mechanistic analysis.

• Reproducibility and Purity: Recombinant production ensures high purity and biological activity, minimizing variability and contamination risks compared to native protein isolation.

In summary, recombinant human GCP-2 is a versatile tool for immunology, microbiology, tissue engineering, and disease modeling, offering reliable activity and experimental control for diverse research applications.

Yes, recombinant human GCP-2 can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity, its concentration is accurately determined, and it is compatible with your assay system.

Key considerations and supporting details:

• Recombinant proteins are commonly used as standards in ELISA assays for cytokines and chemokines, including GCP-2 (CXCL6). Commercial ELISA kits for human GCP-2 typically use recombinant human GCP-2 as the standard to generate the calibration curve.
• The standard curve is essential for quantifying unknown samples in ELISA, and the accuracy of quantification depends on the quality and precise concentration of the standard protein.
• It is important to ensure that the recombinant GCP-2 standard is fully biologically active and structurally similar to the native protein, as this affects antibody recognition and assay performance.
• The standard should be prepared and diluted according to best practices to avoid technical errors that could compromise quantification. Follow the same buffer and matrix conditions as your samples whenever possible.
• If you are developing your own ELISA (not using a commercial kit), you must validate that your recombinant GCP-2 standard produces a reliable and linear standard curve within the assay’s dynamic range.

Summary of best practices:

• Use a recombinant GCP-2 standard of known concentration and high purity.
• Prepare the standard curve in the same matrix as your samples if possible.
• Validate the standard curve for linearity and reproducibility.
• Ensure the recombinant standard is recognized by the antibodies used in your ELISA.

If you are using a commercial ELISA kit, it is generally recommended to use the standard provided with the kit, as it has been validated for that specific assay. However, if you are developing your own assay or need to replace the standard, a well-characterized recombinant human GCP-2 is appropriate for calibration.

Recombinant Human GCP-2 (CXCL6) has been validated for several applications in published research, primarily in the fields of immunology, microbiology, and cartilage biology.

Key validated applications include:

• Antibacterial Activity Assays: Recombinant human GCP-2 has been shown to possess direct antibacterial activity against both Gram-positive (e.g., Staphylococcus aureus, Streptococcus pyogenes) and Gram-negative (e.g., Escherichia coli, Pseudomonas aeruginosa) bacteria. These activities were validated using bactericidal assays, membrane disruption studies, and fluorescence microscopy to visualize peptide-bacteria interactions.

• Chemotaxis Assays: GCP-2 is a potent chemoattractant for neutrophils. Its chemotactic activity has been validated in vitro by measuring neutrophil migration in response to recombinant protein, typically using transwell or Boyden chamber assays.

• Angiogenesis Assays: GCP-2 has been implicated in angiogenesis, and its effects have been validated in endothelial cell migration and tube formation assays, although these are less frequently reported than chemotaxis or antibacterial assays.

• Cartilage and Chondrogenesis Studies: Recombinant GCP-2 has been used to stimulate chondrocytes and enhance extracellular matrix (ECM) formation in human cartilage organoids and in vivo models. Applications include supplementation of cartilage implants and intra-articular injection in animal models of osteoarthritis, with outcomes assessed by histological staining (e.g., Toluidine Blue, collagen type II immunostaining), micro-CT, and biochemical assays for ECM components.

• Western Blot and Binding Studies: The binding of GCP-2 to bacterial surfaces has been validated by Western blot analysis, confirming its interaction with microbial membranes.

• Signal Transduction Assays: GCP-2-induced activation of intracellular signaling pathways (e.g., AKT phosphorylation) in chondrocytes and other cell types has been validated by immunoblotting and related biochemical assays.

• In Vivo Disease Models: Recombinant GCP-2 and its mutants have been used in animal models (e.g., mouse models of osteoarthritis) to assess effects on inflammation, pain, and cartilage integrity, with validation by histopathology and behavioral assays.

Summary Table of Validated Applications

These applications are supported by peer-reviewed studies and are widely used in both basic and translational research on chemokines and tissue regeneration.

Reconstitution Protocol

Initial Preparation

Begin by allowing the lyophilized GCP-2 protein vial to reach room temperature before opening. Centrifuge the vial prior to opening to ensure all lyophilized material is at the bottom. Reconstitute the protein in sterile distilled water or an aqueous buffer containing 0.1% bovine serum albumin (BSA) to achieve a concentration of 0.1–1.0 mg/mL. Alternatively, sterile 18 MΩ·cm water can be used at a minimum concentration of 100 µg/mL, which can then be further diluted to desired concentrations.

Reconstitution Procedure

Allow the vial to reconstitute for 15–30 minutes at room temperature with gentle agitation. Avoid vigorous shaking, as this can cause foaming and potentially denature the protein. If the protein is supplied in lyophilized form, gentle mixing is essential to ensure complete dissolution without introducing air bubbles that could compromise protein stability.

Storage and Stability

Short-term Storage

After aseptic reconstitution, store the protein at 2–8°C for up to one month under sterile conditions. This storage condition is suitable for experiments with a 5–7 day cycle, such as induced maturation of dendritic cells. During the experimental period, remove only the required amount from refrigeration and add it directly to culture medium.

Long-term Storage

For extended storage, maintain the reconstituted protein at −20 to −70°C under sterile conditions for up to three months. For even longer-term storage, add 5–50% glycerol (final concentration) as a cryoprotectant and aliquot the protein before freezing at −20 to −80°C. The lyophilized protein itself remains stable for at least two years when stored at −20°C from the date of receipt.

Important Considerations

Avoid repeated freeze-thaw cycles, as these can reduce protein activity and stability. When preparing aliquots for long-term storage, consider diluting the reconstituted protein with carrier protein-containing solutions such as 0.1% BSA, 10% fetal bovine serum (FBS), or 5% human serum albumin (HSA) before freezing. This approach protects the protein from denaturation during storage and handling.