Recombinant Rat MIP-3α

Recombinant Rat MIP-3α (also known as CCL20) is a chemokine widely used in research to study immune cell recruitment, mucosal immunity, inflammation, and related signaling pathways. Its primary value lies in its ability to selectively attract dendritic cells (DCs), T cells, and mesenchymal stem cells (MSCs), making it a powerful tool for dissecting immune mechanisms in vitro and in vivo.

Key scientific applications and advantages include:

• Chemoattractant Activity: Recombinant MIP-3α is a potent chemoattractant for CD34⁺-derived dendritic cells and T lymphocytes, but not for monocytes or neutrophils. This specificity allows precise modeling of immune cell migration and tissue infiltration in response to inflammatory or infectious stimuli.

• Mucosal Immunity and Lymphoid Tissue Formation: MIP-3α is crucial for the formation and function of mucosal lymphoid tissues by directing lymphocytes and DCs toward epithelial sites, making it essential for studies on mucosal immunology, gut-associated lymphoid tissue, and barrier immunity.

• Modeling Inflammation and Disease: MIP-3α is upregulated in inflamed and mucosal tissues and plays a role in recruiting immune cells during inflammation, infection, and tissue injury. Recombinant protein can be used to mimic these processes in animal models or cell-based assays.

• Enhancing Vaccine and Immunotherapy Studies: In vivo, MIP-3α can recruit immature DCs to sites of antigen delivery, enhancing antigen presentation and the induction of adaptive immune responses, particularly cytotoxic T lymphocyte (CTL) responses. This makes it valuable for vaccine adjuvant research and cancer immunotherapy models.

• Cell Migration and Chemotaxis Assays: Recombinant MIP-3α is commonly used in transwell migration assays to quantify the chemotactic response of various immune cell subsets, including MSCs and DCs.

• Receptor Specificity: MIP-3α signals primarily through the CCR6 receptor, enabling targeted studies of CCR6-mediated pathways and their roles in immunity and disease.

• Standardization and Reproducibility: Using recombinant protein ensures batch-to-batch consistency, defined activity, and the absence of confounding factors present in tissue extracts or conditioned media, which is critical for quantitative and mechanistic studies.

In summary, recombinant rat MIP-3α is a versatile reagent for immunological research, particularly for dissecting mechanisms of immune cell trafficking, mucosal immunity, inflammation, and for enhancing the efficacy of immunotherapies and vaccines in preclinical models.

Yes, you can use recombinant rat MIP-3α as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to your assay system.

Supporting details:

• Recombinant proteins are commonly used as ELISA standards: Many commercial rat MIP-3α ELISA kits include recombinant rat MIP-3α as the standard for generating the calibration curve, allowing quantification of both natural and recombinant forms in samples.
• Purity and quantification: For accurate calibration, the recombinant protein should be highly purified and its concentration precisely determined, ideally by methods such as HPLC or spectrophotometry.
• Validation: It is essential to validate that your recombinant standard is immunologically equivalent to the native protein in your samples. This ensures that the antibodies in your ELISA recognize the recombinant form with the same affinity as the endogenous protein. Validation can be performed by spike-and-recovery experiments and parallelism testing.
• Lot-to-lot variability: Be aware that different lots of recombinant protein may show slight differences in immunoreactivity, which can affect quantification. Assigning values based on ELISA measurement rather than relying solely on the mass stated on the vial is recommended for quality control.
• Standard curve preparation: Follow best practices for preparing ELISA standards, including careful reconstitution, dilution, and handling to minimize errors.

Best practices:

• Use the recombinant rat MIP-3α standard at concentrations spanning the expected range in your samples.
• Validate the standard curve for linearity, accuracy, and reproducibility.
• Confirm that the recombinant protein is recognized by the antibodies used in your ELISA.

Summary: Recombinant rat MIP-3α is suitable as a standard for ELISA quantification, provided it is properly validated and prepared according to best practices for ELISA calibration.

Recombinant Rat MIP-3α (CCL20) has been validated in published research primarily for applications involving chemotaxis assays, studies of inflammatory responses, and investigation of its role in immune cell recruitment and antiviral activity.

Key validated applications include:

• Chemotaxis Assays: Recombinant MIP-3α has been used to demonstrate potent chemoattractant activity for T lymphocytes and dendritic cells (DCs), particularly in transwell migration or Boyden chamber assays. These assays establish its functional role in directing immune cell migration.

• Receptor Characterization: Studies have used recombinant MIP-3α to identify and characterize its specific receptor (CCR6) on target cells, often employing calcium mobilization assays in transfected cell lines (e.g., HEK293 cells expressing candidate receptors).

• Inflammation and Disease Models: Recombinant MIP-3α has been applied in animal models and ex vivo systems to investigate its role in inflammatory cascades, such as in ischemic brain injury, where it was shown to be pivotal in mediating leukocyte infiltration and inflammatory responses.

• Antiviral Activity: Recombinant MIP-3α has been validated for its ability to inhibit viral replication in vitro, such as demonstrating dose-dependent antiviral effects against vaccinia virus in plaque reduction assays.

• Expression and Detection Studies: While not a direct application of the recombinant protein itself, antibodies against MIP-3α (validated in WB, IHC, and ELISA) are used to detect its expression in tissues and cell lysates, supporting studies of its biological distribution and regulation.

• Cellular and Tissue Culture: Recombinant MIP-3α is used as a standard or stimulant in cell culture experiments to study immune cell activation, migration, and cytokine production.

Summary Table: Validated Applications for Recombinant Rat MIP-3α

Additional Notes:

• Most functional studies use recombinant MIP-3α at physiologically relevant concentrations to mimic in vivo conditions.
• While some studies focus on human or mouse MIP-3α, the rat protein is used in analogous assays due to high sequence and functional conservation.
• The protein is often used in both carrier-free and BSA-containing formulations, depending on the application (e.g., cell culture vs. ELISA standard).

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

Reconstitution Protocol

Reconstitute lyophilized recombinant rat MIP-3α by first equilibrating both the vial and reconstitution buffer to room temperature. Centrifuge the vial to concentrate the lyophilized powder at the bottom of the tube. The protein should be reconstituted at a concentration of 200 µg/mL in sterile phosphate-buffered saline (PBS). Add the appropriate volume of sterile PBS containing at least 0.1% human serum albumin or bovine serum albumin as a carrier protein. Allow the reconstitution to proceed for 15-30 minutes with gentle agitation. If flakes remain visible, continue mixing for approximately 2 hours at room temperature.

Storage Conditions

Short-term storage: After reconstitution, store the protein solution at 2°C to 8°C for up to one month.

Long-term storage: For extended storage, freeze working aliquots at -20°C to -70°C in a manual defrost freezer. The inclusion of carrier protein (0.1% human serum albumin or bovine serum albumin) is recommended for long-term stability.

Critical consideration: Avoid repeated freeze-thaw cycles, as these can compromise protein integrity and biological activity.

Preparation for Cell Culture

Prepare a stock solution of no less than 25 µg/mL from the reconstituted protein. For specific cell culture applications, further dilute the stock solution to the desired working concentration using sterile PBS or your appropriate cell culture medium. The biological activity of recombinant rat MIP-3α can be verified through chemotaxis assays or other functional assays specific to your experimental design.

Ensure all reconstitution and handling procedures maintain sterility to prevent contamination and preserve protein functionality throughout your experiments.