Recombinant Human CCL14a/HCC-1

Recombinant Human CCL14a/HCC-1 is a chemokine widely used in research for its roles in immune cell migration, modulation of inflammatory responses, and potential anti-viral and anti-tumor activities.

Key scientific reasons to use recombinant CCL14a/HCC-1 in research applications:

• Chemotaxis Assays: CCL14a/HCC-1 induces the migration of T lymphocytes, monocytes, and eosinophils, making it valuable for studying immune cell trafficking, inflammation, and tissue infiltration mechanisms.
• Receptor Biology: It is a ligand for CCR1, CCR3, and CCR5 chemokine receptors, which are important in immune signaling and are also implicated in HIV infection and other pathologies.
• Anti-HIV Research: CCL14a/HCC-1 exhibits anti-HIV activity by interacting with CCR5, a co-receptor for HIV entry, and can inhibit infection by M-tropic HIV-1 strains.
• Cancer Biology: Altered CCL14 expression is associated with tumor progression and immune infiltration in several cancers. Overexpression of CCL14 can reduce cell migration, invasion, and proliferation in cancer cell lines, and is correlated with better prognosis and immune cell infiltration in tumor microenvironments.
• Stem Cell and Hematopoiesis Studies: CCL14a/HCC-1 can enhance proliferation of CD34+ myeloid progenitor cells, relevant for studies on hematopoiesis and stem cell biology.
• Cell Signaling: It induces intracellular calcium changes and enzyme release in monocytes, useful for dissecting chemokine signaling pathways.
• Protein-Protein Interaction and Immunology: Recombinant CCL14a/HCC-1 is used in protein-protein interaction studies, immunological assays, and cell biology experiments to elucidate chemokine function and signaling.

Typical applications include:

• Chemotaxis and migration assays
• Receptor binding and signaling studies
• Immune cell activation and differentiation experiments
• Cancer cell proliferation and invasion assays
• Anti-viral mechanism research
• Biomarker validation and quantification in clinical samples

When using recombinant CCL14a/HCC-1, ensure proper controls and validation of biological activity, as the full-length protein and its processed forms may differ in potency and receptor specificity. This protein is for laboratory research only and not for diagnostic or therapeutic use.

Yes, recombinant human CCL14a/HCC-1 can be used as a standard for quantification or calibration in ELISA assays, provided it is validated for this purpose and matches the form of CCL14a detected by your assay.

Supporting details:

• ELISA kits for CCL14a/HCC-1 routinely use recombinant human CCL14a as the standard to generate the calibration curve for quantification of CCL14a in biological samples. The standard curve is essential for determining the concentration of CCL14a in unknown samples by comparing their signal to that of known concentrations of recombinant protein.
• Both natural and recombinant CCL14a are recognized by validated ELISA kits, and the recombinant standard is specifically included for quantitative measurement. The assay’s antibodies are designed to detect both forms equivalently, assuming the recombinant protein is properly folded and processed.
• The standard must be of the same isoform and sequence as the analyte detected by your assay. CCL14a exists in different processed forms (e.g., full-length or mature/secreted), so ensure your recombinant standard matches the form recognized by your ELISA antibodies.
• Validation and documentation: Commercial ELISA kits provide rigorous validation data showing that recombinant CCL14a standards yield accurate, linear, and reproducible calibration curves. If you are using a recombinant standard outside of a kit, you should validate its performance in your specific assay setup.

Best practices:

• Reconstitute and store the recombinant standard according to the manufacturer’s instructions to maintain stability and activity.
• Prepare a fresh standard curve for each assay to account for any variability in handling or storage.
• If using a custom or in-house recombinant CCL14a, confirm its purity, concentration, and biological activity, and verify that it is recognized by your assay antibodies.

In summary: Recombinant human CCL14a/HCC-1 is the standard of choice for ELISA quantification, as long as it is validated for your assay and matches the detected analyte form.

Recombinant Human CCL14a/HCC-1 has been validated in published research for several key applications, primarily in the fields of immunology, cell biology, and cancer research.

Validated Applications:

• Chemotaxis Assays:
  CCL14a/HCC-1 is widely used to study chemotaxis (directed cell migration) of immune cells, including T lymphocytes, monocytes, and eosinophils. Recombinant CCL14a/HCC-1 has been shown to induce chemotaxis in these cell types, and its activity can be neutralized by specific antibodies, confirming its functional role in migration assays.

• HIV Infection Inhibition Studies:
  The protein has been used to demonstrate inhibition of M-tropic HIV-1 infection in vitro, highlighting its relevance in virology and immunology research.

• Protein-Protein Interaction Analyses:
  Recombinant CCL14a/HCC-1 is employed in protein-protein interaction studies, particularly to investigate its interactions with chemokine receptors such as CCR1, CCR3, and CCR5, which are relevant for immune cell signaling and migration.

• Cell Biology and Functional Studies:
  The protein is used in cell proliferation, migration, and invasion assays, especially in cancer research. For example, studies have used CCL14 overexpression or recombinant protein to assess its effects on tumor cell migration, invasion, and proliferation, as well as its role in modulating the tumor immune microenvironment.

• Biomarker and Expression Analysis:
  CCL14 has been investigated as a biomarker in various cancers, including lung adenocarcinoma, hepatocellular carcinoma, ovarian, and thyroid cancers. Research includes expression analysis (e.g., qPCR, ELISA) and correlation with immune cell infiltration and patient prognosis.

Additional Context:

• Neutralization Assays:
  Recombinant CCL14a/HCC-1 is used in neutralization assays to confirm the specificity of antibodies or to block its biological activity in functional studies.

• Therapeutic Development:
  The protein is also utilized in therapeutic development research, particularly for its immunomodulatory properties and potential as a target in cancer and infectious disease.

Summary Table of Validated Applications

These applications are supported by both primary research articles and product validation data, confirming the broad utility of recombinant human CCL14a/HCC-1 in experimental and translational research.

To reconstitute and prepare Recombinant Human CCL14a/HCC-1 protein for cell culture experiments, follow these best practices based on available protocols and manufacturer recommendations:

Reconstitution Steps

1. Centrifuge the Vial:
   Briefly centrifuge the lyophilized protein vial before opening to ensure all powder is at the bottom.

2. Reconstitution Solution:
   Reconstitute the protein in sterile distilled water (or 18MΩ-cm H₂O for higher purity). Some protocols suggest using an aqueous buffer with 0.1% BSA to stabilize the protein, especially if you plan to store aliquots.

3. Concentration:
   Reconstitute to a concentration between 0.1–1.0 mg/mL (commonly 0.1–0.5 mg/mL is recommended). For example, add 100–1000 µL of sterile water to 100 µg of protein.

4. Mixing:
   Gently mix by pipetting up and down or allow the vial to sit at room temperature for 15–30 minutes with gentle agitation. Avoid vortexing or vigorous pipetting to prevent protein denaturation.

Preparation for Cell Culture

• Further Dilution:
  Dilute the reconstituted protein in cell culture medium or buffer to the desired working concentration. Typical working concentrations for chemokines in cell culture range from 1–100 ng/mL, depending on the experiment.

• Aliquoting:
  Prepare small working aliquots to minimize freeze-thaw cycles. Store aliquots at –20°C to –80°C for long-term storage.

• Stability:
  After reconstitution, the protein is stable for up to 1 month at 2–8°C under sterile conditions, or 6 months at –20°C to –80°C.

Additional Notes

• Sterility:
  Always use sterile technique to avoid contamination, especially for cell culture applications.

• Buffer Considerations:
  If the protein is sensitive to aggregation or loss of activity, consider including a carrier protein (e.g., 0.1% BSA) in the reconstitution buffer.

• Activity Testing:
  Validate the biological activity of the reconstituted protein in your specific cell culture system (e.g., chemotaxis assay, migration assay).

Summary Protocol

1. Centrifuge vial.
2. Reconstitute with sterile distilled water (or 0.1% BSA in water) to 0.1–1.0 mg/mL.
3. Gently mix; do not vortex.
4. Aliquot and store at –20°C or –80°C.
5. Dilute in cell culture medium to working concentration (e.g., 1–100 ng/mL).
6. Use sterile technique throughout.

This approach ensures optimal protein stability and activity for cell culture experiments.