Recombinant Human CCL16

Recombinant Human CCL16 is a valuable tool for research applications focused on immunology, cancer biology, and inflammation due to its well-characterized chemokine activity, involvement in disease progression, and ability to modulate immune cell behavior.

Key scientific reasons to use recombinant CCL16:

• Chemotactic Activity: CCL16 selectively attracts monocytes and lymphocytes, but not neutrophils, making it useful for studying cell migration, chemotaxis assays, and immune cell recruitment.
• Immune Modulation: It enhances macrophage effector and antigen-presenting functions, and augments T cell cytolytic activity, which is critical for research on immune responses and tumor immunology.
• Cancer Research: CCL16 is implicated in tumor progression, especially in hepatocellular carcinoma (HCC), where it modulates macrophage polarization, promotes angiogenesis, and correlates with poor prognosis. Recombinant CCL16 enables mechanistic studies and therapeutic target validation in cancer models.
• Inflammation and Disease Models: CCL16 is associated with various inflammatory conditions (e.g., ulcerative colitis, cardiovascular disease, chronic kidney disorders), making it relevant for modeling and dissecting inflammatory pathways.
• Receptor Interaction Studies: CCL16 interacts with CCR1, CCR2, CCR5, and CCR8, allowing for detailed analysis of chemokine-receptor signaling and downstream effects in vitro and in vivo.
• Structural and Biophysical Research: Recombinant CCL16, with its unique C-terminal extension, is suitable for structural biology studies to understand chemokine domain function and glycosaminoglycan binding.

Typical applications include:

• Chemotaxis and migration assays
• Immune cell activation and polarization studies
• Tumor microenvironment modeling
• ELISA and binding assays for receptor-ligand interactions
• In vivo disease models (e.g., xenograft studies, fibrosis, inflammation)

Best practices:
Use recombinant CCL16 in controlled, dose-dependent experiments to dissect its specific effects on cell populations, signaling pathways, and disease phenotypes. Confirm activity with appropriate positive and negative controls, and consider its receptor specificity when designing assays.

Summary:
Recombinant Human CCL16 is essential for mechanistic studies of immune cell trafficking, tumor biology, and inflammatory diseases, providing a reproducible and well-defined reagent for diverse experimental protocols.

Yes, you can use recombinant human CCL16 as a standard for quantification or calibration in your ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common practice in quantitative ELISA protocols, where a standard curve is generated using known concentrations of the target protein, often in recombinant form.

Key considerations and best practices:

• Purity and Quantification: The recombinant CCL16 used as a standard should be highly purified, and its concentration should be accurately measured, ideally by an orthogonal method such as HPLC or absorbance at 280 nm with a known extinction coefficient.
• Standard Curve Preparation: Prepare a serial dilution of the recombinant CCL16 to generate a standard curve covering the expected range of your samples (commonly 0–1000 pg/mL, but this can be adjusted as needed).
• Matrix Matching: For best accuracy, dilute the recombinant standard in the same buffer or matrix as your samples (e.g., serum, plasma, or cell culture medium) to minimize matrix effects.
• Validation: If you are not using the standard provided with a commercial ELISA kit, you should validate that your recombinant CCL16 is recognized equivalently by the assay antibodies. This can be done by comparing the standard curve generated with your recombinant protein to that of the kit standard, if available.
• Lot-to-Lot Variation: Be aware that different lots of recombinant protein may have slight differences in immunoreactivity or mass determination, which can affect quantification. It is recommended to value-assign the mass based on measurement in the ELISA itself, rather than relying solely on the mass stated on the vial.
• Species and Isoform Specificity: Ensure that the recombinant CCL16 matches the species and isoform targeted by your ELISA antibodies, as cross-reactivity or epitope differences can affect quantification accuracy.

Limitations:

• If using a recombinant standard not specifically validated for your ELISA kit, results may differ slightly from those obtained with the kit’s original standard due to differences in protein folding, post-translational modifications, or epitope presentation.
• For regulatory or clinical applications, always follow the kit manufacturer’s recommendations regarding standards and calibration.

Summary Table:

Using recombinant human CCL16 as a standard is scientifically valid and widely practiced, but careful validation and standardization are essential for accurate quantification.

Recombinant Human CCL16 has been validated for several applications in published research, primarily including ELISA, Western blot, functional (chemotaxis) assays, and immunohistochemistry (IHC).

Key validated applications and supporting details:

• ELISA (Enzyme-Linked Immunosorbent Assay): Used for quantifying CCL16 protein levels in biological samples, including plasma and tissue lysates. This is a standard application for chemokine detection and quantification in both clinical and research settings.

• Western Blot: Applied to detect and confirm the presence and molecular weight of recombinant CCL16 in cell lysates or purified protein preparations.

• Functional Assays (Chemotaxis): Recombinant CCL16 has been shown to induce chemotaxis of human monocytes and THP-1 cells, but not resting lymphocytes or neutrophils, confirming its biological activity in vitro. Published studies have also used recombinant CCL16 to assess its ability to recruit immune cells and modulate cell migration in various models.

• Immunohistochemistry (IHC): Used to detect CCL16 protein expression in tissue sections, particularly in studies investigating its role in hepatocellular carcinoma (HCC) and tumor microenvironments. IHC has been employed to correlate CCL16 expression with other markers such as IL-6 and VEGFA in tumor tissues.

Additional research applications include:

• In vivo models: Recombinant CCL16 has been used in xenograft models to study its effects on tumor growth, immune cell recruitment, and cytokine expression in the tumor microenvironment.
• Receptor binding and signaling studies: CCL16 has been validated in assays examining its interaction with chemokine receptors (CCR1, CCR2, CCR5, CCR8) and downstream signaling pathways relevant to inflammation and cancer.
• Cellular assays: Used to study effects on macrophage polarization, angiogenesis, and immune modulation in cancer and inflammatory disease models.

In summary, recombinant human CCL16 is validated for ELISA, Western blot, functional chemotaxis assays, IHC, and in vivo/ex vivo models to study its biological roles in immunity, inflammation, and cancer.

To reconstitute and prepare Recombinant Human CCL16 protein for cell culture experiments, dissolve the lyophilized protein in sterile deionized water to achieve a final concentration between 0.1–1.0 mg/mL.

Essential steps and best practices:

• Reconstitution:

  • Add sterile deionized water directly to the lyophilized protein. Do not vortex; gently pipette or swirl to dissolve.
  • For example, to prepare 1 mg/mL, add 1 mL water per 1 mg protein. For 0.1 mg/mL, add 1 mL water per 0.1 mg protein.
  • Avoid concentrations below 100 μg/mL to maintain protein stability.

• Stabilization (optional but recommended):

  • Add a carrier protein (e.g., 0.1% BSA) or glycerol (5–50% final concentration) to improve stability, especially for long-term storage or repeated freeze-thaw cycles.
  • Carrier proteins help prevent adsorption to tube walls and aggregation.

• Aliquoting and Storage:

  • After reconstitution, aliquot the solution to minimize freeze-thaw cycles.
  • Store aliquots at –20°C to –80°C for long-term use; short-term storage at 4°C is possible for up to 1 month if a carrier protein is present.
  • Avoid repeated freeze/thaw cycles to preserve protein activity.

• Preparation for cell culture:

  • Before use, dilute the stock solution into your cell culture medium to the desired working concentration.
  • Ensure the final buffer conditions (e.g., pH, osmolarity) are compatible with your cell type and experimental design.

Additional notes:

• If the protein was lyophilized from a solution containing organic solvents (e.g., acetonitrile, TFA), ensure complete removal or sufficient dilution before adding to cells.
• Always consult the specific product datasheet for any unique instructions regarding buffer composition or reconstitution, as some preparations may differ.

Summary of key steps:

• Dissolve in sterile water (0.1–1.0 mg/mL).
• Add carrier protein or glycerol if needed.
• Aliquot and store appropriately.
• Dilute into cell culture medium for experiments.

This protocol ensures optimal solubility, stability, and biological activity of recombinant CCL16 for cell-based assays.