Recombinant Human RANTES

Using Recombinant Human RANTES (CCL5) in research is essential for studying immune cell recruitment, inflammation, cancer, tissue regeneration, and HIV infection mechanisms. This chemokine is a well-characterized tool for dissecting cellular signaling, migration, and immune modulation in controlled experimental systems.

Key reasons to use recombinant human RANTES in research applications:

• Immune Cell Recruitment and Inflammation: RANTES is a potent chemoattractant for T cells, eosinophils, basophils, and monocytes, making it critical for modeling and quantifying leukocyte migration and inflammatory responses in vitro and in vivo.
• Cancer and Tumor Microenvironment Studies: RANTES is implicated in tumor progression, metastasis, and the modulation of the tumor microenvironment, particularly through its effects on immune cell infiltration and angiogenesis.
• Tissue Regeneration and Angiogenesis: RANTES promotes endothelial progenitor cell migration, vascular network formation, and muscle regeneration, making it valuable for studies on tissue repair and revascularization after injury.
• HIV Research: RANTES is a natural ligand for CCR5, a co-receptor for HIV-1 entry into cells. It can inhibit HIV-1 infection by blocking CCR5, and recombinant RANTES or its analogs are used to study viral entry and resistance mechanisms.
• Cytokine and Chemokine Signaling: RANTES modulates the expression of other cytokines (e.g., IL-2, IL-5, IL-6, IL-8, TNFα, IL-10), providing a model to study complex cytokine networks and immune regulation.
• Standardization and Reproducibility: Recombinant proteins offer high purity, defined activity, and batch-to-batch consistency, which are essential for reproducible and interpretable experimental results.

Common research applications include:

• Chemotaxis and migration assays to quantify immune cell movement.
• Bioassays for receptor activation and downstream signaling.
• ELISA and immunoassays as a standard or positive control.
• Functional studies in models of inflammation, cancer, tissue injury, and infection.

In summary, recombinant human RANTES is a versatile and reliable reagent for dissecting immune mechanisms, modeling disease processes, and developing therapeutic strategies targeting chemokine pathways.

Recombinant Human RANTES can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to your assay system. Recombinant RANTES is widely used as a calibrator in commercial ELISA kits for quantitative measurement of both natural and recombinant RANTES in biological samples.

Key considerations and supporting details:

• Validation: Commercial ELISA kits routinely use recombinant human RANTES as the standard, and these standards are often calibrated against international reference preparations such as the NIBSC recombinant RANTES standard 95/520. This ensures traceability and accuracy in quantification.

• Parallelism: Studies and kit documentation indicate that standard curves generated with recombinant RANTES are parallel to those obtained with natural RANTES, supporting its use for quantification in biological samples.

• Recovery and Precision: Recovery experiments show high accuracy (typically 95–105%) when recombinant RANTES is spiked into serum, plasma, and cell culture media, indicating that it behaves similarly to endogenous RANTES in the assay context.

• Assay Compatibility: Recombinant RANTES is suitable for use in sandwich ELISA formats, and is compatible with a variety of sample types including serum, plasma (EDTA, heparin, citrate), urine, and cell culture supernatants.

• Preparation: For best results, prepare a standard curve using serial dilutions of recombinant RANTES in the same buffer or matrix as your samples to minimize matrix effects. Ensure the protein is fully solubilized and handled according to best practices for protein standards.

• Documentation: Refer to your specific ELISA kit protocol for recommended concentrations, diluents, and calibration procedures. Most kits provide detailed instructions for preparing and using recombinant RANTES standards.

Summary Table: Use of Recombinant Human RANTES as ELISA Standard

Best Practices:

• Use recombinant RANTES that is well-characterized and, if possible, traceable to an international standard.
• Validate parallelism and recovery in your specific assay system if using a new batch or source.
• Prepare standards in the same matrix as your samples for optimal accuracy.

Conclusion:
Recombinant Human RANTES is scientifically appropriate and widely accepted as a standard for ELISA quantification and calibration, provided assay-specific validation and proper preparation are followed.

Recombinant Human RANTES (CCL5) has been validated for a wide range of applications in published research, including:

• Bioassay (e.g., cell migration, chemotaxis, cytokine expression modulation)
• ELISA (Standard)
• Functional Assay (e.g., chemotaxis, receptor binding)
• Western Blot
• Immunohistochemistry
• Binding Assay / Protein-protein interaction
• Cell Culture
• Surface Plasmon Resonance Microscopy
• SDS-PAGE
• Fluorescence single-molecule counting assays
• In Vivo studies (e.g., animal models of inflammation, cancer, HIV infection)
• Microglia/macrophage and glioma progression studies
• Cancer cell migration and metastasis assays
• HIV entry inhibition studies
• Cytokine and chemokine profiling

These applications are supported by multiple peer-reviewed publications and product validation data from various suppliers.

To properly reconstitute and prepare Recombinant Human RANTES (CCL5) protein for cell culture experiments, follow these best practices based on manufacturer guidelines and scientific protocols:

Reconstitution Steps

1. Centrifuge the Vial:
   Briefly centrifuge the lyophilized protein vial at low speed (3,000–3,500 rpm) for 5 minutes to ensure all powder is at the bottom.

2. Reconstitution Buffer:
   Reconstitute the lyophilized RANTES in sterile PBS (phosphate-buffered saline) or sterile water (18 MΩ-cm H₂O), as recommended by most suppliers.

   • Typical reconstitution concentration: 100 µg/mL (e.g., add 0.1 mL for 10 µg protein).
   • For ELISA standards, some protocols recommend reconstitution to 20–50 ng/mL or 10,000 pg/mL, depending on the kit.

3. Dissolution Technique:

   • Gently pipette the buffer down the side of the vial to avoid foaming.
   • Do not vortex—this can denature the protein.
   • Allow the vial to sit at room temperature for 15–30 minutes with gentle agitation (e.g., rocking or gentle pipetting) until fully dissolved.

4. Carrier Protein (Optional but Recommended):
   For cell culture applications, consider adding a carrier protein (e.g., 0.1–1% BSA, 10% FBS, or 5% HSA) to the reconstitution or dilution buffer to prevent protein loss due to adsorption to surfaces.

Dilution for Cell Culture

• After reconstitution, dilute the protein to the desired working concentration using cell culture medium or PBS containing carrier protein (e.g., 0.1% BSA or 10% FBS).
• Typical working concentrations for bioassays range from 1–100 ng/mL, depending on the experiment.

Storage and Handling

• Short-term: Store reconstituted protein at 2–8°C for up to one week if used immediately.
• Long-term: Aliquot and store at –20°C to –80°C in a manual defrost freezer. Avoid repeated freeze-thaw cycles.
• For ELISA standards: Use immediately after reconstitution or freeze aliquots at –20°C or –80°C for later use.

Summary Protocol

1. Centrifuge vial (3,000–3,500 rpm, 5 min).
2. Reconstitute in sterile PBS or water to 100 µg/mL (or as specified).
3. Gently mix, do not vortex.
4. Dilute in culture medium or PBS with carrier protein to working concentration.
5. Aliquot and store at –20°C or –80°C for long-term use.
6. Avoid repeated freeze-thaw cycles.

This approach ensures optimal protein stability and activity for cell culture experiments.