Recombinant Mouse RANTES

Using Recombinant Mouse RANTES (CCL5) in research applications is essential for studying immune cell recruitment, inflammation, tissue regeneration, and disease models in a controlled and reproducible manner. Recombinant RANTES allows precise manipulation of chemokine signaling in mouse systems, enabling mechanistic studies and therapeutic investigations relevant to immunology, neuroscience, oncology, and regenerative medicine.

Key scientific reasons to use recombinant mouse RANTES include:

• Immune Cell Recruitment: RANTES is a potent chemoattractant for T cells, monocytes, eosinophils, basophils, and memory T helper cells, making it critical for dissecting leukocyte migration and immune response mechanisms in vitro and in vivo.
• Inflammation and Disease Modeling: It plays a central role in inflammatory diseases such as asthma, autoimmune disorders, and infection models, allowing researchers to mimic or modulate inflammatory responses in mouse models.
• Neuroscience Applications: RANTES is involved in neuronal restoration, axonogenesis, and protection of hippocampal memory function after brain injury, making it valuable for neuroinflammation and neuroregeneration studies.
• Cancer and Tumor Microenvironment: RANTES influences tumor growth, angiogenesis, and immune cell infiltration in the tumor microenvironment, supporting studies on cancer immunology and metastasis.
• Angiogenesis and Tissue Repair: Recombinant RANTES promotes angiogenesis and muscle regeneration, as demonstrated in mouse models of ischemia and tissue injury.
• Receptor and Signaling Studies: As a natural ligand for CCR1, CCR3, and CCR5, recombinant RANTES is used to study chemokine-receptor interactions, downstream signaling, and receptor-specific functions.
• Standardization and Reproducibility: Recombinant proteins provide batch-to-batch consistency, defined concentration, and purity, which are critical for reproducible experimental results and quantitative assays such as ELISA, bioassays, and cell migration studies.

Common research applications include:

• Chemotaxis and migration assays
• In vivo inflammation and tissue injury models
• Bioassays for receptor activation or inhibition
• ELISA standards and controls
• Mechanistic studies of immune cell function and signaling pathways

Using recombinant mouse RANTES ensures species specificity, which is crucial for mouse-based experiments due to potential differences in chemokine-receptor interactions between species. This enables accurate modeling of mouse immune and disease processes, supporting translational research and preclinical studies.

Yes, recombinant mouse RANTES can be used as a standard for quantification or calibration in ELISA assays, provided it is highly purified and its concentration is accurately known. This approach is widely accepted in research applications for quantifying RANTES in biological samples.

Supporting details:

• ELISA kits for mouse RANTES commonly use recombinant mouse RANTES as the standard. These kits demonstrate that recombinant RANTES yields standard curves that are parallel to those obtained with natural RANTES, allowing for reliable quantification of both recombinant and endogenous protein in samples.
• Calibration and quantification: The recombinant standard should be serially diluted to generate a standard curve covering the expected concentration range in your samples. The concentration of RANTES in unknown samples is then interpolated from this curve.
• Purity and quantification: It is essential that the recombinant RANTES is highly purified and its concentration is accurately determined, typically by absorbance at 280 nm or amino acid analysis, to ensure reliable calibration.
• Matrix effects: When preparing standards, dilute the recombinant protein in the same buffer or matrix as your samples (e.g., assay diluent, serum, or culture medium) to minimize matrix effects and ensure accurate quantification.
• Species specificity: Ensure that the antibodies in your ELISA are specific for mouse RANTES and that the recombinant protein matches the species and isoform being measured.

Best practices:

• Use freshly prepared or properly stored aliquots of recombinant RANTES to avoid degradation.
• Validate the standard curve in your assay system, especially if using a custom or in-house ELISA, to confirm linearity and parallelism with endogenous RANTES in your sample matrix.
• Avoid mixing standards or reagents from different sources or lots, as this can introduce variability.

Limitations:

• Recombinant proteins expressed in different systems (e.g., E. coli vs. mammalian cells) may have different post-translational modifications, which could affect antibody recognition in rare cases. However, most commercial ELISA kits are validated for recombinant standards.
• For diagnostic applications, additional validation may be required, but for research quantification, recombinant mouse RANTES is appropriate and widely used.

In summary: Recombinant mouse RANTES is suitable and commonly used as a standard for ELISA quantification, provided it is pure, accurately quantified, and matched to the assay system.

Recombinant Mouse RANTES (CCL5) has been validated for several key applications in published research, primarily in the fields of immunology, neuroscience, and cancer biology. The most commonly validated applications include:

• Bioassays: Used to measure chemotactic activity, especially the ability to attract immune cells such as BaF3 mouse pro-B cells transfected with human CCR5, macrophages, and other leukocytes.
• In Vivo Studies: Applied in animal models to investigate roles in neuroprotection, memory function, brain injury recovery, ischemia therapy, kidney injury, and cancer immune control.
• Neutralization Assays: Used to study the blocking or modulation of CCL5 activity in vivo, particularly in the context of memory formation and synaptic function.
• Binding Assays: Employed to assess ligand-receptor interactions, including studies on chemokine-binding proteins and receptor specificity.
• Functional Assays: Validated for use in functional studies of immune cell recruitment, inflammation, and tissue injury.
• ELISA Standards: Used as a standard in enzyme-linked immunosorbent assays for quantifying CCL5/RANTES levels.
• Western Blotting and Immunohistochemistry: Utilized for detection and quantification of CCL5 in tissue and cell lysates, often in conjunction with specific antibodies.

Supporting details and representative research applications:

• Neuroscience: CCL5 has been shown to be essential for axonogenesis and neuronal restoration after brain injury, and for protecting hippocampal memory function following mild traumatic brain injury in mouse models.
• Immunology and Inflammation: RANTES is widely used to study leukocyte recruitment to inflammatory sites, including T cells, macrophages, eosinophils, and basophils. It is also implicated in the pathogenesis of autoimmune and neuroinflammatory diseases.
• Cancer Biology: Validated in studies examining tumor microenvironment modulation, cancer immune control, and bone metastasis.
• Kidney Injury: Used in models of acute kidney injury to study immune cell recruitment and tissue protection.
• Ischemia and Angiogenesis: Applied in mouse ischemia therapy to evaluate pro-angiogenic effects.
• Cell Migration and Chemotaxis: Frequently used in transwell migration assays and other chemotaxis assays to validate its role in immune cell movement.

Summary Table of Validated Applications

Key insights:

• Recombinant Mouse RANTES is a versatile tool validated for both in vitro and in vivo research, especially for studying immune cell chemotaxis, neuroprotection, inflammation, and cancer biology.
• Its use spans bioassays, animal models, neutralization, binding, and functional assays, as well as standardization in ELISA and detection in immunoassays.

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

To reconstitute and prepare Recombinant Mouse RANTES (CCL5) protein for cell culture experiments, follow these steps for optimal solubility, stability, and bioactivity:

1. Centrifuge the vial:
Before opening, briefly centrifuge the lyophilized protein vial to ensure all material is at the bottom.

2. Reconstitution buffer:

• Use sterile PBS (phosphate-buffered saline) as the primary reconstitution buffer.
• If the protein formulation contains no carrier protein (e.g., BSA or trehalose), or for sensitive applications, add 0.1% BSA (bovine serum albumin) to the PBS to stabilize the protein and prevent adsorption to plastic surfaces.
• Alternatively, sterile distilled water can be used if recommended by the datasheet, but PBS is preferred for cell culture compatibility.

3. Reconstitution concentration:

• Reconstitute to a stock concentration of 100 μg/mL in sterile PBS (with or without 0.1% BSA, depending on your application and the protein formulation).
• For other working concentrations, adjust the volume accordingly using the formula:
  [\text{Volume to add (μL)} = \frac{\text{Amount of protein (μg)}}{\text{Desired concentration (μg/μL)}}]

4. Dissolving the protein:

• Gently pipette up and down or swirl the vial to dissolve the protein.
• Do not vortex or shake vigorously to avoid denaturation or foaming.
• Allow the protein to fully dissolve at room temperature for 15–30 minutes with gentle agitation.

5. Aliquot and storage:

• Once fully dissolved, aliquot the stock solution into small 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 week), store at 4°C.
• Avoid repeated freeze-thaw cycles, as this can reduce protein activity.

6. Working solution preparation:

• Dilute the stock solution to the desired working concentration in your cell culture medium immediately before use.
• If using serum-free media, ensure the presence of a carrier protein (e.g., BSA) to prevent loss of activity due to adsorption.

7. General notes:

• Always use sterile technique to prevent contamination.
• Confirm the absence of endotoxin if your application is sensitive to it.
• Check the specific datasheet for any protein-specific recommendations, as formulations may vary.

Summary Table: Key Steps for Recombinant Mouse RANTES Reconstitution

These steps will ensure your recombinant mouse RANTES is properly prepared for reliable and reproducible cell culture experiments.