Recombinant Mouse RANK Ligand

Recombinant Mouse RANK Ligand (RANKL) is widely used in research applications because it is a critical regulator of osteoclast differentiation, bone metabolism, immune cell function, and tissue development in mouse models.

Key scientific reasons to use recombinant mouse RANKL include:

• Osteoclastogenesis and Bone Research: RANKL is essential for the formation, activation, and survival of osteoclasts, the cells responsible for bone resorption. In vitro, recombinant mouse RANKL is routinely used to induce differentiation of monocytes or bone marrow-derived macrophages into osteoclasts, especially when combined with M-CSF. This is fundamental for studying bone remodeling, osteoporosis, arthritis, and other bone-related diseases.

• Immune System Studies: RANKL-RANK signaling is crucial for lymph node formation, thymic microenvironment establishment, and dendritic cell survival. Recombinant mouse RANKL enables investigation of immune cell activation, T cell development, and thymic regeneration, particularly in aging or immunodeficient models.

• Cell Signaling and Functional Assays: RANKL is a member of the TNF superfamily and activates multiple signaling pathways, including NFATc1 and NF-κB, which are central to osteoclast-specific gene transcription and immune responses. Recombinant RANKL is used in cell culture to dissect these pathways and their downstream effects.

• Disease Modeling and Therapeutic Research: Manipulating RANKL levels in mouse models allows researchers to mimic pathological conditions such as osteoporosis, rheumatoid arthritis, cancer metastasis to bone, and inflammatory bone loss. It is also used to test pharmacological inhibitors or antibodies targeting the RANKL-RANK-OPG axis.

• Thymic Regeneration and Aging: Exogenous recombinant RANKL administration has been shown to restore thymic architecture and function in aged mice, improving T cell responses to vaccination and tumors, making it valuable for aging and regenerative studies.

Typical applications for recombinant mouse RANKL include:

• In vitro differentiation of osteoclasts from precursor cells.
• Functional assays of RANKL-RANK signaling in bone and immune cells.
• Modeling bone diseases and testing therapeutic interventions.
• Studying immune cell development, activation, and thymic regeneration.

Best practices: Use recombinant mouse RANKL at experimentally determined concentrations (often 0.5–2 ng/mL for osteoclast differentiation), ensure high purity and low endotoxin levels, and validate biological activity in your specific assay system.

In summary, recombinant mouse RANKL is indispensable for mechanistic studies of bone biology, immune regulation, and disease modeling in mouse systems due to its central role in osteoclastogenesis and immune cell function.

You can use recombinant mouse RANK Ligand (RANKL) 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 for cytokines and other proteins.

Key considerations and supporting details:

• Recombinant RANKL is routinely used as a standard in commercial mouse RANKL ELISA kits, which are designed to quantify both natural and recombinant forms of the protein. These kits typically include a lyophilized recombinant RANKL standard for generating the calibration curve.
• Purity and formulation matter: For best results, use a highly purified recombinant protein. Some protocols recommend using a formulation with BSA (bovine serum albumin) as a carrier for stability, especially for ELISA standards. Carrier-free formulations are also available and may be preferable if BSA could interfere with your assay.
• Concentration accuracy: The recombinant standard must have a precisely known concentration, ideally determined by a reliable method such as HPLC or absorbance at 280 nm with an accurate extinction coefficient.
• Standard curve preparation: Prepare a serial dilution of the recombinant RANKL to generate a standard curve covering the expected range of your samples. This allows for accurate interpolation of unknown sample concentrations.
• Validation: Commercial ELISA kits validate their quantification using recombinant standards and demonstrate linearity and sensitivity across a defined range. If you are developing your own assay, validate that your recombinant standard produces a linear, reproducible standard curve in your assay conditions.

Limitations and best practices:

• Ensure the recombinant RANKL matches the isoform and epitope recognized by the antibodies in your ELISA, especially if you are assembling a custom assay.
• Avoid using recombinant proteins labeled for bioassay use as ELISA standards unless the manufacturer specifically states they are suitable for ELISA calibration.
• Always run the standard curve in parallel with your samples in each assay to account for any day-to-day or batch-to-batch variation.

In summary, recombinant mouse RANKL is appropriate and widely used as a standard for ELISA quantification, provided it is pure, accurately quantified, and compatible with your assay system.

Recombinant Mouse RANK Ligand (RANKL) has been validated in published research for applications including osteoclast differentiation assays, cell signaling studies, immune cell activation, tumor biology, and as a standard or control in biochemical assays.

Key validated applications include:

• Osteoclast differentiation assays: RANKL is widely used to induce differentiation of monocytes/macrophages (e.g., RAW 264.7 cells, bone marrow-derived macrophages) into osteoclasts in vitro, often in combination with M-CSF. This is the most common application, enabling studies of bone resorption, osteoporosis, and osteoclast biology.

• Cell signaling and functional assays: RANKL is used to study signaling pathways in the TNF receptor family, including activation of NFATc1 and downstream gene transcription in osteoclast precursors. It is also used to investigate the role of RANKL-RANK interactions in immune cell activation and differentiation.

• Bioassays and ELISA standards: Recombinant RANKL serves as a standard or control in ELISA and Western blot assays, and is used in neutralization experiments to assess the activity of inhibitors or antibodies targeting the RANKL-RANK pathway.

• Tumor biology and immune modulation: RANKL has been used to study its effects on tumor growth, osteolytic bone metastases, and immune cell function, including dendritic cell survival and T cell growth.

• Thymic and immune system research: RANKL is involved in thymic epithelial cell development and immune reconstitution studies, particularly in models of transplantation and thymic defects.

• Bone metabolism and disease models: RANKL is validated in models of osteoporosis, bone loss, and related diseases, including as an immunogen for generating anti-RANKL antibodies and in studies of bone homeostasis.

• Other biochemical and cell biology applications: RANKL is used in cell culture, mass spectrometry, HPLC, SDS-PAGE, cell adhesion, cell proliferation, and immunohistochemistry protocols.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and product validation data, confirming the utility of recombinant mouse RANKL in diverse research areas related to bone biology, immunology, and cell signaling.

Reconstitution Procedures

Recombinant Mouse RANK Ligand (RANKL) proteins are typically supplied in lyophilized form and require proper reconstitution before use in cell culture experiments. The standard reconstitution concentration is 0.1 to 1.0 mg/mL in sterile water or appropriate buffer solutions. For example, reconstituting 100 µg of protein requires between 100 µL and 1 mL of diluent to achieve the desired concentration range.

Most protocols recommend reconstituting at a concentration of 0.1 mg/mL as a starting point, which can then be further diluted in aqueous solutions as needed for specific experimental applications. Some formulations specify reconstitution at 50 µg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA).

Handling and Storage Considerations

Critical handling steps include the following:

When reconstituting the lyophilized protein, allow several minutes for complete reconstitution and do not vortex the solution, as this can cause protein denaturation. A quick spin of the vial is recommended before adding the diluent.

For formulations containing carrier proteins, stock solutions can be prepared at 50–100 µg/mL in appropriate sterile buffer with 0.2–1% BSA or human serum albumin (HSA) added when preparing working solutions. This carrier protein addition is particularly important for preventing protein loss through adsorption to container surfaces.

Storage and Stability

Short-term storage of reconstituted RANKL should be maintained at 4°C for 2–7 days. For long-term storage, the protein should be stored below −18°C or at −80°C. When preparing aliquots for prolonged storage, dilute the protein to working concentrations in a 0.1% BSA solution and store at −80°C.

The lyophilized protein itself is stable for six to twelve months when stored desiccated at −20°C to −70°C. Avoid repeated freeze-thaw cycles, as these can compromise protein activity and stability.

Biological Activity Considerations

When reconstituted properly, recombinant Mouse RANKL maintains its biological activity for inducing osteoclast differentiation. The expected ED₅₀ (effective dose at 50% response) for osteoclast differentiation of mouse splenocytes is typically 0.5–2 ng/mL, though some formulations report ED₅₀ values of 5–15 ng/mL in the presence of cross-linking antibodies. Endotoxin levels should be verified as less than 0.01 EU/µg to ensure suitability for cell culture applications.