Recombinant Human Soluble RANK Ligand

Recombinant Human Soluble RANK Ligand (sRANKL) is widely used in research applications because it is a critical cytokine for inducing and studying osteoclast differentiation, activation, and survival, as well as for investigating bone metabolism, immune regulation, and related disease mechanisms.

Key scientific reasons to use recombinant human sRANKL include:

• Osteoclastogenesis Studies: sRANKL is essential for the in vitro differentiation of monocytes or precursor cells into osteoclasts, especially when combined with M-CSF. This is fundamental for modeling bone resorption, studying osteoclast biology, and screening anti-resorptive drugs.
• Bone Remodeling Research: The RANKL/RANK/OPG signaling axis is central to bone modeling and remodeling. Disruption or modulation of this pathway is implicated in osteoporosis, arthritis, cancer metastases to bone, and other bone diseases.
• Immunology and Lymphoid Organ Development: RANKL is involved in lymph node formation, thymic microenvironment establishment, dendritic cell survival, and B cell maturation, making it valuable for immunological studies.
• Cancer and Metastasis Models: RANKL/RANK signaling is a target in research on tumor bone metastasis and may be involved in tumor cell proliferation, making sRANKL useful for investigating cancer biology and potential therapies.
• Functional Assays and Drug Screening: Recombinant sRANKL enables controlled, reproducible functional assays for testing inhibitors, antibodies, or small molecules targeting the RANKL/RANK pathway.
• Therapeutic Development: Engineered or mutant forms of RANKL are being explored as therapeutic agents or vaccines for osteoporosis and other bone-related diseases, demonstrating the translational relevance of sRANKL in preclinical models.

Best practices for using recombinant human sRANKL:

• Use in combination with M-CSF for robust osteoclast differentiation.
• Validate biological activity by assessing osteoclast formation or matrix degradation in vitro.
• Employ appropriate controls and dose-response experiments to ensure specificity and reproducibility.

In summary: Recombinant human soluble RANK Ligand is indispensable for research into bone biology, osteoclast function, immune system development, and related pathologies due to its central role in the RANKL/RANK/OPG signaling pathway.

Yes, you can use Recombinant Human Soluble RANK Ligand (RANKL) as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is purified and its concentration is accurately determined.

Key Points for Using Recombinant RANKL as an ELISA Standard:

• Purified Protein: The recombinant RANKL should be highly purified to ensure accurate calibration. Impurities can affect the standard curve and lead to inaccurate quantification.
• Concentration Determination: The concentration of the recombinant protein should be measured precisely, ideally using methods such as amino acid analysis, HPLC, or spectrophotometry.
• Compatibility: Ensure that the recombinant RANKL is compatible with the antibodies used in your ELISA kit. Most commercial ELISA kits are calibrated against recombinant RANKL, so using a recombinant standard is standard practice.
• Standard Curve: Prepare a serial dilution of the recombinant RANKL to generate a standard curve. This curve will be used to interpolate the concentration of RANKL in your samples.

References:

• Biomedica ELISA Kit: This kit is calibrated against human recombinant RANKL protein, indicating that recombinant RANKL is suitable for use as a standard.
• Abcam ELISA Kit: The kit uses a recombinant RANKL standard for calibration.
• R&D Systems DuoSet ELISA: Includes a recombinant standard for use in the assay.

Best Practices:

• Storage: Store the recombinant RANKL according to the manufacturer's instructions to maintain its stability and activity.
• Validation: Validate the standard curve by running known concentrations of the recombinant RANKL and ensuring that the curve is linear and reproducible.

By following these guidelines, you can effectively use Recombinant Human Soluble RANK Ligand as a standard for your ELISA assays.

Recombinant Human Soluble RANK Ligand has been validated for a range of applications in published research, primarily in the fields of bone biology, immunology, and cell signaling. The most commonly validated applications include:

• In vitro differentiation of monocytes to osteoclasts: RANKL, in combination with M-CSF, is routinely used to induce osteoclastogenesis from human or mouse monocytes or precursor cells in culture. This is the gold-standard assay for studying osteoclast formation and function.

• Bioactivity assays: RANKL is validated for use in bioassays to test its ability to activate RANK signaling, induce osteoclast differentiation, and stimulate downstream pathways such as c-Jun N-terminal kinase activation.

• ELISA standard: Recombinant RANKL is used as a standard in ELISA assays to quantify RANKL levels in biological samples.

• Western blot control: It serves as a positive control for Western blotting to detect RANKL or to validate antibody specificity.

• Neutralization assays: Used to assess the efficacy of neutralizing antibodies or small molecules that inhibit RANKL-RANK interactions, often in the context of drug discovery for osteoporosis or bone metastasis.

• Functional studies in immune cell activation and dendritic cell survival: RANKL is used to study its effects on dendritic cell survival, T cell-dependent immune responses, and broader immune modulation.

• Cell-receptor signaling studies: RANKL is applied to dissect signaling pathways downstream of RANK, including in cancer, osteogenesis, and inflammation research.

• Surface Plasmon Resonance (SPR): Used to characterize binding kinetics between RANKL and its receptor or inhibitors.

Additional validated research applications include:

• Investigation of lymph node organogenesis and thymic microenvironment establishment.
• Study of mammary gland development.
• Matrix degradation and inflammation modulation.
• Drug screening and structure-based inhibitor development: Used in both in vitro and in vivo models to validate small molecule inhibitors targeting RANKL-RANK interactions, especially for anti-osteoporotic therapies.

Summary Table of Validated Applications

These applications are widely supported by both product validation data and peer-reviewed research, making recombinant human soluble RANK Ligand a versatile tool in bone, immune, and cancer biology.

To reconstitute and prepare Recombinant Human Soluble RANK Ligand (sRANKL) for cell culture experiments, dissolve the lyophilized protein in sterile water at a concentration of 0.1 mg/mL (100 µg/mL), then dilute further in cell culture medium containing a carrier protein such as 0.1% BSA, 5% HSA, or 5–10% FBS to prevent adsorption and degradation.

Step-by-step protocol:

• Preparation:

  • Briefly centrifuge the vial to collect the powder at the bottom before opening.
  • Open the vial carefully to avoid contamination.

• Reconstitution:

  • Add sterile water (or as specified in the datasheet) along the inner wall of the vial to reach the desired concentration (typically 0.1 mg/mL).
  • Gently pipet up and down to dissolve the protein. Do not vortex, as vigorous mixing may reduce biological activity.
  • Allow the solution to sit at room temperature for 20 minutes to ensure complete dissolution.
  • The solution should be clear; if not, continue gentle mixing.

• Dilution for cell culture:

  • Dilute the reconstituted stock into cell culture medium containing a carrier protein (e.g., 0.1% BSA, 5% HSA, or 5–10% FBS) to reach your working concentration.
  • Avoid diluting directly into water for cell culture use, as this may cause protein degradation.

• Aliquoting and storage:

  • Prepare aliquots to avoid repeated freeze-thaw cycles.
  • Store aliquots at −20°C to −80°C with carrier protein for long-term stability.
  • For short-term use (up to 1 month), storage at 2–8°C is acceptable.

Additional notes:

• Always consult the specific product datasheet for buffer composition and recommended reconstitution conditions, as some preparations may contain stabilizers or require alternative buffers.
• For functional assays, typical working concentrations for sRANKL range from 5–50 ng/mL, but optimization may be required for your cell type and experimental design.
• Avoid repeated freeze-thaw cycles, which can denature the protein and reduce activity.

Summary Table:

This protocol ensures optimal solubility, stability, and biological activity of recombinant human soluble RANK Ligand for cell culture experiments.