Recombinant Human CCL28

Recombinant Human CCL28 is used in research applications to study and manipulate processes such as angiogenesis, immune cell recruitment, wound healing, and antimicrobial defense. Its recombinant form allows for controlled, reproducible experiments on its biological functions and signaling pathways.

Key scientific applications and rationale include:

• Angiogenesis and Vascular Biology: CCL28, via its receptor CCR10, activates signaling pathways (eNOS/NO, Src, PI3K, MAPK) in human endothelial cells, promoting endothelial proliferation, survival, and tube formation—key steps in new blood vessel formation. Recombinant CCL28 has been shown to enhance angiogenesis in vitro and in vivo, making it valuable for studying vascular development, tissue repair, and tumor biology.

• Wound Healing: CCL28/CCR10 signaling regulates dermal microvessel angiogenesis and improves skin wound healing, as demonstrated in mouse models. Recombinant CCL28 can be used to dissect these mechanisms or test therapeutic strategies for impaired healing.

• Cardiac and Ischemic Tissue Repair: In models of myocardial and hindlimb ischemia, recombinant CCL28 promotes angiogenesis and tissue repair, suggesting its utility in cardiovascular research and regenerative medicine.

• Immunology and Mucosal Defense: CCL28 is constitutively expressed by mucosal epithelial cells and recruits immune cells (e.g., IgA-secreting plasma cells) to mucosal surfaces, supporting studies on mucosal immunity, inflammation, and host-pathogen interactions.

• Antimicrobial Activity: CCL28 exhibits direct antimicrobial effects, including anti-Candida activity, making recombinant CCL28 useful for investigating innate immune mechanisms and developing novel antimicrobial agents.

• Mechanistic Studies: Using recombinant protein allows precise control over concentration and timing, enabling detailed analysis of CCL28’s effects on cell signaling, gene expression, and cellular behavior in various human and animal cell types.

• Therapeutic Target Validation: Recombinant CCL28 is essential for preclinical studies evaluating its potential as a therapeutic agent or target in diseases involving angiogenesis, chronic wounds, cardiovascular disease, or mucosal infections.

In summary, recombinant human CCL28 is a versatile tool for dissecting its roles in angiogenesis, immune regulation, tissue repair, and antimicrobial defense, and for validating therapeutic strategies targeting these pathways.

Yes, recombinant human CCL28 can be used as a standard for quantification or calibration in ELISA assays, provided it is of sufficient purity and accurately quantified. Recombinant proteins are commonly used as standards in quantitative ELISA protocols for chemokines such as CCL28.

Key considerations for using recombinant CCL28 as an ELISA standard:

• Purity and Quantification: The recombinant CCL28 should be highly purified and its concentration accurately determined, ideally by methods such as HPLC or UV spectrophotometry. Impurities or inaccurate quantification can compromise the reliability of your standard curve.

• Formulation: Standards are often supplied lyophilized and must be reconstituted according to the manufacturer’s instructions. Carrier-free formulations are preferred for ELISA calibration, as carriers (e.g., BSA) may interfere with assay sensitivity or specificity.

• Validation: The recombinant CCL28 should be validated for use in ELISA. Many commercial ELISA kits for CCL28 include recombinant CCL28 as the standard, demonstrating its suitability for this purpose. If using a custom recombinant preparation, ensure it is compatible with your assay’s antibodies and detection system.

• Standard Curve Preparation: Prepare a serial dilution of the recombinant CCL28 to generate a standard curve covering the expected concentration range in your samples (typically from low pg/mL to several ng/mL). The standard curve should be run in parallel with your samples for accurate quantification.

• Matrix Effects: If your samples are complex (e.g., serum, plasma), matrix effects may influence quantification. It is advisable to prepare standards in a matrix similar to your samples or use appropriate diluents to minimize these effects.

Protocol Example:

1. Reconstitute recombinant CCL28 according to instructions.
2. Prepare serial dilutions in assay buffer or sample matrix.
3. Add standards and samples to ELISA plate coated with anti-CCL28 antibody.
4. Proceed with detection and quantification as per your ELISA protocol.

Limitations:

• Recombinant standards are not recommended for bioassays unless specifically validated for biological activity.
• Lot-to-lot variability and protein stability should be monitored.

Summary: Recombinant human CCL28 is suitable as a standard for ELISA quantification if it is pure, accurately quantified, and validated for your assay system. Always follow best practices for standard preparation and assay optimization to ensure reliable results.

Recombinant Human CCL28 has been validated in published research for several key applications, primarily in functional studies related to immunology, antimicrobial activity, and angiogenesis.

Validated Applications in Published Research:

• Antimicrobial Activity Assays:
  Recombinant human CCL28 has been shown to possess potent antimicrobial activity, particularly against Candida albicans and other fungi, as well as gram-positive and gram-negative bacteria. Studies have used recombinant CCL28 in both in vitro and in vivo infection models to demonstrate rapid and sustained fungicidal activity, membrane disruption, and therapeutic potential for oropharyngeal candidiasis.

• Angiogenesis Assays (In Vitro and In Vivo):
  CCL28 has been validated in in vitro assays for endothelial cell tube formation, migration, and proliferation. In vivo, recombinant CCL28 has been used in Matrigel plug assays in mice and chick chorioallantoic membrane (CAM) assays to demonstrate its ability to promote neovascularization and angiogenesis. These studies have established its role in tumor angiogenesis and tissue repair, including cardiac and wound healing models.

• Cell Migration and Chemotaxis Assays:
  Recombinant CCL28 has been used to study the migration of IgA-expressing cells and lymphocytes to mucosal tissues, as well as the recruitment of regulatory T cells, via chemotaxis and adhesion assays.

• Immunomodulation Studies:
  Research has utilized recombinant CCL28 to investigate its role in bridging innate and adaptive immunity, including its effects on B and T cell homing and function.

• Protein Detection and Quantification:
  Recombinant CCL28 is commonly used as a standard or positive control in ELISA, Western blot, SDS-PAGE, and protein array applications to detect and quantify CCL28 in biological samples.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and reviews, confirming the utility of recombinant human CCL28 in diverse experimental systems.

To reconstitute Recombinant Human CCL28 protein for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS) to a final concentration of 100 μg/mL. For optimal results, follow these steps:

• Briefly centrifuge the vial to collect the powder at the bottom before opening.
• Add sterile PBS directly to the vial to achieve the desired concentration (e.g., 100 μg/mL).
• Gently mix by swirling or inverting; avoid vigorous shaking to prevent foaming and protein denaturation.
• Allow the solution to sit at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.

Additional best practices:

• If a different buffer is specified in your product datasheet (e.g., 20 mM phosphate buffer, pH 7.4, 130 mM NaCl), use that buffer for reconstitution.
• For long-term storage, aliquot the reconstituted protein and store at –20°C to –70°C. Avoid repeated freeze-thaw cycles to maintain protein integrity.
• For short-term use, store at 2–8°C for up to one month.
• If your cell culture protocol requires lower working concentrations, dilute the stock solution further in cell culture medium or buffer immediately before use.

General notes:

• Always use sterile technique to prevent contamination.
• If the protein is to be used in sensitive assays or cell culture, consider adding a carrier protein (e.g., 0.1% BSA) to minimize adsorption and loss, unless contraindicated for your application.
• Confirm the absence of endotoxin if required for your cell type or experiment.

These steps ensure that Recombinant Human CCL28 is properly prepared for cell culture applications, maintaining its biological activity and stability.