Recombinant Mouse MIP-1γ

Recombinant mouse MIP-1γ (macrophage inflammatory protein 1 gamma, also designated CCL9/10) is a valuable tool for immunological and inflammatory research due to its well-characterized biological functions and versatile applications.

Biological Significance

MIP-1γ is a CC chemokine with established roles in immune regulation and inflammation. This protein mediates critical immune functions including the migration and recruitment of CD4+ and CD8+ T cells, as well as neutrophil chemoattraction. The chemokine is selectively produced by Th1 cells and plays a role in maintaining and amplifying Th1-type immune responses through CCR1 receptor signaling. Additionally, MIP-1γ plays an important role in osteoclast differentiation and survival, making it relevant for bone biology research.

Research Applications

Immune Cell Migration Studies: The recombinant protein can be used to investigate chemotactic responses in cell migration assays. Bioactivity is typically measured through the protein's ability to chemoattract cells transfected with CCR1 receptors, with effective dose ranges of 2-10 ng/mL.

Inflammatory Response Modeling: MIP-1γ is useful for studying inflammatory pathways and immune cell recruitment to inflamed or infected tissues. The protein can be applied in models examining Th1-mediated immune responses and cytokine production patterns.

Osteoclastogenesis Research: The chemokine's role in osteoclast activation and survival makes it applicable for bone resorption and bone biology studies.

Bioassay Development: Recombinant MIP-1γ serves as a standard for ELISA and other immunoassays, though bioassay-grade formulations are specifically recommended for functional studies.

Technical Advantages

The recombinant protein is typically supplied as a lyophilized preparation with high purity (>97-98% by SDS-PAGE) and low endotoxin levels (<0.01 EU/µg). The molecular weight is approximately 11.5 kDa. When properly stored at -20°C to -70°C in desiccated conditions, the protein remains stable for 6-12 months. After reconstitution, it can be stored at 2-8°C for one month or at -20°C to -70°C for extended periods. The recombinant protein demonstrates stability through multiple freeze-thaw cycles without significant loss of activity.

Yes, recombinant Mouse MIP-1γ can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately known.

For ELISA quantification:

• Recombinant proteins are commonly used as standards to generate a standard curve, which allows for the quantification of the target analyte in biological samples.
• The recombinant Mouse MIP-1γ should be highly purified (typically >95% purity), and its concentration should be precisely determined, often by absorbance at 280 nm or using a protein assay.
• The standard should be diluted in the same buffer or matrix as your samples to minimize matrix effects and ensure accurate calibration.
• Prepare a serial dilution of the recombinant protein to cover the expected detection range of your assay (e.g., 7.8–500 pg/mL as described in typical ELISA protocols).

Important considerations:

• Verify compatibility: The recombinant standard must match the target detected by your ELISA antibodies (i.e., same isoform, sequence, and post-translational modifications if relevant).
• Use fresh standard solutions: Prepare the standard immediately before use, as recommended in ELISA protocols, to avoid degradation or loss of activity.
• Do not use standards for bioassays unless validated: Some recombinant proteins are specifically tested for ELISA calibration but not for functional bioassays.

In summary, using recombinant Mouse MIP-1γ as an ELISA standard is scientifically valid and widely practiced, provided you follow best practices for protein handling, dilution, and assay setup.

Recombinant Mouse MIP-1γ (also known as CCL9/CCL10) has been validated for several applications in published research, primarily in the context of its biological activity as a chemokine and its quantification in biological samples.

Validated Applications:

• Chemotaxis Assays:
  The most direct and widely validated application is the use of recombinant Mouse MIP-1γ in in vitro chemotaxis assays. Its biological activity is confirmed by its ability to chemoattract mouse BaF/3 cells transfected with human CCR1 and human THP-1 cells, with reported ED₅₀ values in the low nanogram per milliliter range. This demonstrates its functional role in recruiting immune cells such as T cells, monocytes, and dendritic cells.

• Bioassays for Functional Characterization:
  Recombinant Mouse MIP-1γ is used in bioassays to study its effects on various cell types, including suppression of colony formation by murine and human bone marrow myeloid progenitor cells and regulation of bone resorption. It is also used to investigate its induction by RANKL during osteoclastogenesis and by TLR-9 activation in microglia.

• Multiplex Immunoassays (e.g., LEGENDplex):
  MIP-1γ has been validated as a quantifiable analyte in multiplex bead-based immunoassays for mouse serum and plasma, allowing simultaneous measurement of multiple chemokines in a single sample. This application is important for profiling inflammatory responses in mouse models.

• In Vivo Studies of Inflammation and Immunity:
  Recombinant MIP-1γ has been used in animal models to study its role in immune cell recruitment, inflammation, and bone metabolism. For example, it has been implicated in the recruitment of CD11b⁺ dendritic cells to Peyer’s patches and in the regulation of osteoclast differentiation and survival.

Supporting Context:

• Disease Models:
  MIP-1γ is studied in the context of inflammatory diseases, bone metabolism disorders, and sepsis. Its expression and function have been analyzed in models of sepsis, where it is measured as a marker of systemic inflammation.

• Mechanistic Studies:
  Research has used recombinant MIP-1γ to dissect signaling pathways involving CCR1 and to study its regulation by nuclear receptors and pattern recognition receptors.

Summary Table:

If you need protocols or further details for a specific application, please specify the experimental context.

To reconstitute and prepare Recombinant Mouse MIP-1γ protein for cell culture experiments, briefly centrifuge the vial to collect the contents, then reconstitute in sterile, distilled water to a concentration between 0.1–1.0 mg/mL. For working solutions and further dilutions, use aqueous buffers supplemented with a carrier protein such as 0.1–1.0% BSA (bovine serum albumin) to stabilize the protein and minimize adsorption to surfaces.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to ensure all lyophilized protein is at the bottom.
• Add sterile, distilled water to achieve your desired stock concentration (typically 0.1–1.0 mg/mL).
• Gently mix by pipetting up and down; do not vortex, as vigorous mixing can denature the protein.
• Aliquot the stock solution to avoid repeated freeze-thaw cycles, which can degrade the protein.
• For working concentrations, dilute the stock in sterile PBS or cell culture medium containing 0.1–1.0% BSA. This carrier protein helps maintain protein stability and bioactivity.
• Store reconstituted protein at 2–8°C for up to one month, or at –20°C to –70°C for longer-term storage. Avoid repeated freeze-thaw cycles.

Additional notes:

• The recommended working concentration for cell-based assays (e.g., chemotaxis) is typically in the range of 0.002–0.6 μg/mL, depending on the assay and cell type.
• Always use aseptic technique to prevent contamination.
• If the protein was lyophilized from a solution containing acetonitrile or TFA, ensure complete evaporation before reconstitution, or consult the product datasheet for specific instructions.
• For best results, prepare fresh working solutions before each experiment.

This protocol ensures optimal solubility, stability, and biological activity of Recombinant Mouse MIP-1γ for cell culture applications.