Recombinant Human MIP-3α

Recombinant Human MIP-3α (also known as CCL20) is widely used in research due to its key roles in immune cell chemotaxis, modulation of inflammatory responses, and its utility in immunological and infectious disease models. Its applications span basic immunology, vaccine development, cancer research, and studies of inflammatory and infectious diseases.

Key scientific reasons to use Recombinant Human MIP-3α in research:

• Chemotaxis of Immune Cells: MIP-3α is a potent chemoattractant for immature dendritic cells (DCs), memory T cells, and monocytes, primarily via the CCR6 receptor. This makes it valuable for studying immune cell migration, tissue homing, and the mechanisms of immune surveillance and inflammation.

• Modeling Inflammatory Diseases: Elevated MIP-3α expression is observed in chronic inflammatory conditions such as rheumatoid arthritis, psoriasis, and atopic dermatitis. Using recombinant MIP-3α allows researchers to model and dissect the molecular pathways involved in these diseases, including the recruitment of immune cells to inflamed tissues.

• Vaccine and Immunotherapy Research: Fusion of MIP-3α to vaccine antigens enhances antigen targeting to immature dendritic cells, resulting in stronger and more sustained antibody and T-cell responses. This approach has been shown to improve vaccine efficacy in preclinical models, including for SARS-CoV-2 and cancer immunotherapy.

• Antimicrobial and Antiviral Studies: MIP-3α exhibits direct antimicrobial activity against bacteria and viruses, and is involved in the innate immune response to pathogens. Recombinant MIP-3α can be used to study these mechanisms or to test its therapeutic potential in infectious disease models.

• Functional Assays: Recombinant MIP-3α is used in chemotaxis assays to quantify the migration of T cells, dendritic cells, or monocytes in response to chemokine gradients. It is also used in colony formation assays to study its inhibitory effects on myeloid progenitor proliferation.

• Mucosal Immunity and Epithelial Biology: MIP-3α is highly expressed in mucosal tissues and is involved in the recruitment of immune cells during infection or inflammation, making it useful for studies of mucosal immunity and epithelial cell biology.

Summary of typical research applications:

• Dissecting immune cell trafficking and chemokine signaling pathways.
• Modeling and analyzing chronic inflammatory and autoimmune diseases.
• Enhancing vaccine antigen delivery and immunogenicity.
• Investigating antimicrobial and antiviral defense mechanisms.
• Performing functional migration and proliferation assays with immune cells.

In summary, Recombinant Human MIP-3α is a versatile tool for immunological research, enabling precise manipulation and analysis of chemokine-driven processes in both health and disease.

Yes, recombinant human MIP-3α (CCL20) can be used as a standard for quantification or calibration in ELISA assays, provided that the recombinant protein is highly purified and its concentration is accurately determined. The standard curve generated with the recombinant protein should be parallel to the standard curve obtained with the kit's native standards to ensure accurate quantification of naturally occurring MIP-3α in your samples.

Key Points:

• Purified Recombinant Protein: Use a purified recombinant human MIP-3α (preferably from a reliable source, such as E. coli-expressed and well-characterized).
• Accurate Concentration: The concentration of the recombinant protein should be determined using a reliable method (e.g., amino acid analysis, HPLC, or spectrophotometry).
• Parallelism: The recombinant protein should generate a standard curve that is parallel to the curve generated with the kit's standards, indicating that the assay recognizes both recombinant and natural forms similarly.
• Kit Compatibility: Most commercial ELISA kits for human MIP-3α (CCL20) are designed to recognize both recombinant and natural forms of the protein, as noted in several kit summaries (e.g., Quantikine, Invitrogen, BioLegend, and others).

Recommendations:

• Always follow the manufacturer's instructions for standard preparation and reconstitution.
• If using a recombinant protein not provided by the kit manufacturer, validate its suitability by comparing the standard curve with the kit's provided standards.
• For best results, use the recombinant protein in the same buffer as the kit's standards to minimize matrix effects.

In summary, recombinant human MIP-3α is suitable for use as a standard in ELISA assays for quantifying MIP-3α, as long as it is properly purified and its concentration is accurately known.

Recombinant Human MIP-3α (CCL20) has been validated in published research for several key applications, primarily as a chemoattractant in cell migration assays, as an immunomodulatory agent in vaccine studies, and for its antimicrobial activity.

Key validated applications include:

• Chemotaxis/Bioassays:
  Recombinant MIP-3α is widely used to assess chemotactic responses of immune cells, particularly immature dendritic cells (DCs), T cells, and CCR6+ cells. Its activity is typically measured by its ability to induce migration of these cells in vitro, often using transwell or Boyden chamber assays. For example, it has been shown to be a potent chemoattractant for certain DCs and T cells, with activity confirmed by concentration-dependent chemotaxis assays using human T cells.

• Immunological Modulation in Vaccine Research:
  MIP-3α has been fused to vaccine antigens to enhance immune responses by targeting antigens to immature dendritic cells. Studies have validated its use in DNA vaccine constructs, where fusion to MIP-3α increases both antibody and T-cell responses, as demonstrated in SARS-CoV-2 and Mycobacterium tuberculosis vaccine models. These studies confirm expression and function of recombinant MIP-3α fusion proteins in both in vitro and in vivo immunization protocols.

• Antimicrobial Activity:
  Recombinant MIP-3α and its C-terminal peptides have been validated in antimicrobial assays, showing direct bactericidal activity against pathogens such as Escherichia coli and Staphylococcus aureus, as well as antiviral and antifungal effects. These assays typically involve incubating the protein or peptide with microbial cultures and assessing viability.

• Inhibition of Myeloid Progenitor Proliferation:
  MIP-3α has been shown to inhibit the proliferation of myeloid progenitors in colony formation assays, indicating a role in hematopoietic regulation.

• Adhesion and Migration Studies:
  It has been used to study the adhesion of memory CD4+ T cells and the transmigration of leukocytes across endothelial barriers, particularly in the context of inflammation and immune cell trafficking.

• Disease Model Studies:
  Recombinant MIP-3α has been applied in models of cancer, rheumatoid arthritis, and infectious diseases to study its role in immune cell recruitment and disease progression.

Summary Table of Validated Applications

Additional Notes:

• Recombinant MIP-3α is typically validated for use in whole cell-based assays and is not generally used for direct detection (e.g., ELISA), though antibodies against MIP-3α are used for such purposes.
• Its activity is mediated through the CCR6 receptor, which is expressed on immature dendritic cells and certain T cell subsets.

If you require protocols or more specific details for a particular application, please specify the context or assay type.

To reconstitute and prepare Recombinant Human MIP-3α (CCL20) for cell culture experiments, dissolve the lyophilized protein in sterile, distilled water to a concentration of 0.1–1.0 mg/mL. After initial reconstitution, make further dilutions in tissue culture-grade buffered solutions containing heat-inactivated fetal calf serum or low endotoxin BSA to stabilize the protein and minimize adsorption.

Step-by-step protocol:

1. Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
2. Add sterile, distilled water directly to the vial to achieve a concentration between 0.1–1.0 mg/mL (e.g., for 100 μg, add 100–1000 μL water).
3. Gently pipette to dissolve the protein completely. Avoid vigorous vortexing to prevent denaturation.
4. Aliquot the stock solution to avoid repeated freeze-thaw cycles, which can degrade the protein.
5. Store the reconstituted stock at 2–8 °C for up to 1 week, or at –20 °C or below for longer-term storage. For long-term storage, add a carrier protein (e.g., 0.1% BSA or HSA) to prevent loss of activity.
6. Prepare working dilutions in cell culture medium or PBS containing 0.1% BSA or heat-inactivated serum immediately before use.

Additional notes:

• Avoid repeated freeze-thaw cycles by aliquoting the stock solution.
• Typical working concentrations for cell-based assays are in the range of 10–50 ng/mL, but optimal concentrations should be determined empirically for your specific application.
• If your protein is supplied with a specific buffer or carrier, follow the manufacturer’s recommendations for reconstitution and dilution.

Summary of key points:

• Use sterile, distilled water for initial reconstitution (0.1–1.0 mg/mL).
• Make further dilutions in buffered solutions with carrier protein (BSA or serum).
• Aliquot and store at appropriate temperatures to maintain stability.
• Avoid freeze-thaw cycles and prepare working solutions fresh.

This protocol ensures maximal recovery, stability, and biological activity of recombinant MIP-3α for cell culture experiments.