Recombinant Human MIP-3β

Recombinant Human MIP-3β (also known as CCL19) is widely used in research to study immune cell migration, lymphoid tissue organization, and inflammatory responses due to its role as a chemokine that specifically attracts T cells, B cells, and dendritic cells via the CCR7 receptor.

Key reasons to use Recombinant Human MIP-3β in research applications:

• Chemotaxis Assays: MIP-3β is a potent chemoattractant for human lymphocytes, including T and B cells, and is commonly used to study cell migration in vitro and in vivo.
• Dendritic Cell and T Cell Function: It is critical for the recruitment and positioning of mature dendritic cells and activated T cells in secondary lymphoid organs, making it valuable for immunology studies focused on antigen presentation and adaptive immune responses.
• CCR7 Ligand Studies: As the primary ligand for CCR7, MIP-3β is essential for dissecting CCR7-mediated signaling pathways, which are central to lymphocyte trafficking and immune surveillance.
• Inflammation and Autoimmunity Models: MIP-3β is involved in inflammatory responses and is linked to autoimmune disease mechanisms, providing a tool for modeling these processes in vitro and in animal models.
• Functional Assays: It is used in bioassays to assess chemokine activity, immune cell activation, and cytokine production, supporting studies in immunotherapy, vaccine development, and basic immunology.

Additional context:

• Species Cross-Reactivity: Recombinant human MIP-3β is active on both human and murine cells, allowing for translational studies between human and mouse models.
• Regulation by Cytokines: Its expression is downregulated by anti-inflammatory cytokines such as IL-10, making it useful for studying cytokine networks and immune regulation.
• Tissue and Disease Relevance: MIP-3β is implicated in the migration of immune cells to sites of inflammation, lymphoid tissues, and tumors, supporting research in cancer immunology and tissue-specific immune responses.

In summary, Recombinant Human MIP-3β is a versatile tool for investigating immune cell migration, lymphoid tissue dynamics, and the molecular mechanisms underlying immune responses and inflammation.

You can use recombinant human MIP-3β 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 and accepted practice in quantitative ELISA protocols.

Key considerations:

• Purity and Formulation: The recombinant protein should be highly purified (typically >95%) and free from contaminants that could interfere with antibody binding or detection. Carrier-free formulations are preferred for ELISA standards unless BSA or other carriers are compatible with your assay system.
• Concentration Determination: The concentration of the recombinant standard must be accurately measured, ideally by absorbance at 280 nm or another validated method, to ensure reliable standard curve generation.
• Standard Curve Preparation: Prepare a serial dilution of the recombinant MIP-3β in the same buffer or diluent used for your samples, following the ELISA kit or assay protocol. This enables accurate interpolation of sample concentrations from the standard curve.
• Compatibility: Confirm that the recombinant MIP-3β is recognized by the capture and detection antibodies used in your ELISA. Most commercial ELISA kits are validated to detect both natural and recombinant forms of the analyte.
• Lot-to-Lot Consistency: If using recombinant protein from different lots or sources, be aware that minor differences in folding or post-translational modifications can affect antibody recognition. Consistency in the standard source is important for reproducibility.

Additional notes:

• Some manufacturers specifically recommend their recombinant MIP-3β proteins for use as ELISA standards.
• Always follow the reconstitution and dilution instructions provided with the recombinant protein or ELISA kit, as these can affect the accuracy of your standard curve.

In summary, recombinant human MIP-3β is suitable as an ELISA standard if it is pure, accurately quantified, and compatible with your assay antibodies and protocol.

Recombinant Human MIP-3β (CCL19) has been validated for multiple applications in published research, primarily in bioassays, cell culture, functional assays, ELISA, Western blot, immunohistochemistry, and SDS-PAGE.

Key validated applications include:

• Bioassays: Used to assess chemotactic activity for human lymphocytes, dendritic cells, and T cells, as well as to study immune cell migration and activation.
• Cell Culture: Applied in studies involving immune cell differentiation, migration, and interaction, including co-culture systems and functional immune assays.
• Functional Assays: Employed to investigate ligand-receptor interactions (notably with CCR7), immune cell signaling, and apoptosis inhibition in dendritic cells.
• ELISA: Utilized as a standard or control protein for quantifying CCL19/MIP-3β levels in biological samples.
• Western Blot: Used as a control or reference protein for antibody validation and detection of MIP-3β in cell lysates.
• Immunohistochemistry: Applied for tissue localization and expression studies of MIP-3β.
• SDS-PAGE: Used for purity assessment and as a control in immunological assays.

Representative published research applications:

• Chemotaxis assays for lymphocytes and dendritic cells.
• Studies of immune cell recruitment in cancer, HIV, and autoimmune disease models.
• Investigation of dendritic cell maturation and T cell activation in immunotherapy research.
• Cell migration and homing assays in lymphoid tissue models.
• Functional ligand studies for CCR7 receptor signaling.

Additional context:

• MIP-3β/CCL19 is frequently used in immunological research, cell biology, and protein-protein interaction analyses.
• It is a standard tool for validating chemokine activity, immune cell migration, and receptor-ligand specificity in both basic and translational studies.

If you require protocol details or specific experimental setups for any of these applications, please specify the intended use.

To reconstitute and prepare Recombinant Human MIP-3β (CCL19) for cell culture experiments, follow these best-practice steps based on current protocols and technical datasheets:

1. Preparation Before Reconstitution

• Centrifuge the vial briefly to ensure all lyophilized protein is at the bottom before opening.
• Allow the vial and reconstitution buffer to equilibrate to room temperature to prevent condensation.

2. Choice of Reconstitution Buffer

• For most cell culture applications, sterile PBS (phosphate-buffered saline) or sterile water can be used. Some protocols recommend adding 0.1% BSA (bovine serum albumin) as a carrier protein to stabilize the protein and prevent adsorption to plastic.
  • If the product contains BSA as a carrier, reconstitute in sterile PBS with at least 0.1% BSA.
  • If the product is carrier-free, reconstitute in sterile PBS or sterile water; adding 0.1% BSA is recommended for stability, especially for storage or low-concentration use.

3. Reconstitution Concentration

• Common reconstitution concentrations are 25–100 μg/mL for working stocks.
  • Example: For a 25 μg vial, add 1 mL of buffer for 25 μg/mL; for a 100 μg vial, add 1 mL for 100 μg/mL.

4. Reconstitution Technique

• Gently add the buffer down the side of the vial to avoid foaming.
• Do not vortex; gently swirl or invert the vial to dissolve the protein.
• Allow several minutes for complete dissolution. If undissolved material remains, let it sit at room temperature for up to 30 minutes with occasional gentle mixing.

5. Aliquoting and Storage

• Once fully dissolved, aliquot the solution into single-use volumes to avoid repeated freeze-thaw cycles.
• Store aliquots at -20°C to -80°C for long-term storage. For short-term use (up to 1 month), store at 2–8°C.
• Avoid repeated freeze-thaw cycles, as this can degrade the protein.

6. Working Solution Preparation

• For cell culture, dilute the reconstituted stock to the desired working concentration using cell culture medium or PBS with 0.1% BSA immediately before use.
• Filter sterilize if necessary, especially if using water for reconstitution and sterility is required.

Summary Table: Key Steps for Recombinant Human MIP-3β Reconstitution

Additional Notes:

• Always consult the specific product datasheet for any unique requirements, as formulations may vary.
• If visible particulates remain after reconstitution, gently mix for up to 2 hours at room temperature.
• For bioassays, optimal working concentrations should be determined empirically for your cell type and assay conditions.

This protocol ensures maximal protein stability and biological activity for cell culture experiments.