Recombinant Mouse RANK Ligand

Recombinant Mouse RANK Ligand (RANKL) is widely used in research to study and manipulate processes involving bone metabolism, osteoclast differentiation, and immune cell regulation in mouse models. It is a key tool for in vitro and in vivo experiments where precise control of RANKL concentration and activity is required.

Key reasons to use recombinant mouse RANKL in research applications:

• Osteoclast Differentiation and Bone Metabolism: RANKL is essential for the differentiation and activation of osteoclasts, the cells responsible for bone resorption. Adding recombinant RANKL to mouse monocyte or macrophage cultures, typically alongside M-CSF, induces robust osteoclastogenesis, enabling studies of bone biology, osteoporosis, arthritis, and related diseases.

• Immune System Studies: RANKL is involved in the regulation of T cell–dendritic cell interactions and thymic function. Exogenous RANKL administration can restore thymic architecture and improve T cell responses in aged mice, making it valuable for immunology research, including studies on immune aging and vaccine responses.

• Cell Signaling and Functional Assays: Recombinant RANKL allows for controlled investigation of RANK–RANKL–OPG signaling pathways, which are central to both bone and immune system regulation. This is critical for dissecting molecular mechanisms and for screening potential therapeutic agents targeting these pathways.

• Disease Modeling and Therapeutic Testing: Recombinant RANKL is used to induce bone loss or osteoclast activation in animal models, facilitating the study of osteoporosis, cancer-induced bone disease, and the testing of anti-resorptive drugs or immunotherapies.

• Reproducibility and Specificity: Using recombinant protein ensures batch-to-batch consistency, defined activity, and species specificity, which are crucial for reproducible and interpretable experimental results.

Typical applications include:

• In vitro osteoclastogenesis assays
• Bone resorption studies
• Immune cell activation and differentiation assays
• In vivo models of bone loss or immune modulation

Summary: Recombinant mouse RANKL is a fundamental reagent for research in bone biology, immunology, and disease modeling, providing precise control over RANKL-mediated signaling in mouse systems.

You can use recombinant Mouse RANK Ligand (RANKL) as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. Recombinant RANKL is commonly used as a standard in commercial mouse RANKL ELISA kits, and these kits are validated to quantify both recombinant and endogenous RANKL in biological samples.

Essential context and best practices:

• Purity and Quantification: The recombinant RANKL should be highly purified (typically >95%) and its concentration should be precisely measured, ideally using methods such as HPLC or spectrophotometry.
• Standard Curve Preparation: Prepare serial dilutions of the recombinant RANKL in the same buffer as your samples or in the standard diluent provided with the ELISA kit to ensure matrix compatibility and accurate quantification.
• Validation: Confirm that the ELISA kit you are using recognizes recombinant RANKL with a dose-response curve similar to that of native RANKL. Most commercial kits are validated for this purpose and provide a lyophilized recombinant RANKL standard for calibration.
• Carrier Proteins: If your recombinant RANKL is supplied with a carrier protein (e.g., BSA), ensure this does not interfere with your assay. Carrier-free formulations are recommended if BSA or other additives could affect detection.
• Research Use: All recombinant standards and ELISA kits are for research use only and not for diagnostic or therapeutic procedures.

Additional relevant information:

• Recombinant RANKL standards are used to generate standard curves, allowing interpolation of sample concentrations based on optical density or other readouts.
• The use of recombinant standards is standard practice in cytokine and ligand quantification ELISAs, including RANKL.
• Always include internal controls and verify assay performance with known concentrations to ensure accuracy and reproducibility.

In summary, recombinant Mouse RANK Ligand is suitable as a standard for ELISA quantification, provided you follow best practices for purity, quantification, and assay validation.

Recombinant Mouse RANK Ligand (RANKL) has been validated in published research for a range of applications, primarily in bone biology, immunology, and cancer studies. The most common and well-established applications include:

• Induction of osteoclast differentiation from precursor cells, especially in vitro using mouse bone marrow-derived macrophages or the RAW 264.7 cell line.
• Functional bioassays to study RANKL-mediated signaling, osteoclastogenesis, and bone resorption.
• Cell culture experiments for differentiation studies, particularly in combination with M-CSF to generate osteoclasts from monocytes.
• ELISA standards and Western blot controls for quantifying or detecting RANKL in various samples.
• Neutralization assays to test inhibitors or antibodies targeting the RANKL-RANK pathway.
• Investigation of immune cell activation and dendritic cell function, as RANKL is involved in T cell growth and dendritic cell survival.
• Studies of cell-receptor signaling within the tumor necrosis factor (TNF) receptor superfamily, including research on tumor growth and metastasis.
• Research on bone-related diseases such as osteoporosis, osteolytic bone metastases, and osteosarcoma, including in vivo animal models.
• Exploration of thymic epithelial cell biology and immune reconstitution, particularly in the context of thymus function and T cell selection.

Supporting details and additional validated uses:

• RANKL is frequently used to induce osteoclastogenesis in vitro, serving as a standard reagent for functional differentiation assays and mechanistic studies of bone resorption.
• It is applied in bioassays to test the effects of small molecules, peptides, or genetic modifications on osteoclast formation and function.
• RANKL has been used in immunization protocols to generate anti-RANKL antibodies for therapeutic research, such as in osteoporosis models.
• It is also utilized in studies of immune modulation, including dendritic cell survival and T cell activation, reflecting its broader role in the immune system.
• Additional applications include cell adhesion, cell proliferation, and immunohistochemistry, as reported in some product reviews and protocols.

Summary Table of Validated Applications

These applications are supported by both product validation data and peer-reviewed research, making recombinant mouse RANKL a standard tool in bone and immune system research.

To reconstitute and prepare Recombinant Mouse RANK Ligand (RANKL) protein for cell culture experiments, follow these best-practice steps:

• Centrifuge the vial briefly before opening to ensure all lyophilized protein is at the bottom.
• Reconstitute the protein in sterile water or sterile PBS. The recommended concentration for stock solutions is typically 0.1 mg/mL (100 µg/mL) or higher. For example, add 100 µL sterile water to 10 µg lyophilized protein for a 0.1 mg/mL solution.
• Gently pipette to dissolve the protein. Do not vortex, as vigorous agitation can denature the protein.
• If the protein is carrier-free (no BSA or HSA), consider adding 0.1–1% BSA or HSA to the buffer to stabilize the protein, especially if you plan to store aliquots for repeated use.
• Allow several minutes for complete dissolution at room temperature.
• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can reduce activity.
• Storage:
  • Short-term (2–7 days): Store at 4°C.
  • Long-term: Store aliquots at –20°C or –80°C, ideally in the presence of carrier protein.
• Working concentration for cell culture: Typical final concentrations for functional assays (e.g., osteoclast differentiation) range from 5–100 ng/mL, but optimal dosing should be determined empirically for your specific application.

Summary Table: Reconstitution and Handling

Additional Notes:

• Always consult the specific product datasheet for any unique instructions, as formulations may vary.
• For cell culture, ensure all solutions are sterile and endotoxin levels are appropriate for your application.
• If using for sensitive cell types or in vivo, confirm the absence of preservatives or additives that may affect your experiment.

These steps will ensure optimal solubility, stability, and biological activity of recombinant mouse RANKL for cell culture experiments.