Recombinant Human RANK

Using Recombinant Human RANK in research applications is essential for studying the RANK/RANKL/OPG signaling pathway, which is central to bone metabolism, osteoclastogenesis, immune regulation, and certain pathological conditions such as osteoporosis, arthritis, and cancer metastasis to bone.

Key reasons to use recombinant human RANK include:

• Mechanistic Studies of Osteoclastogenesis: RANK is the receptor for RANKL (Receptor Activator of Nuclear Factor κB Ligand), and their interaction is critical for the differentiation, activation, and survival of osteoclasts—the cells responsible for bone resorption. Recombinant human RANK enables in vitro reconstitution of this pathway, allowing precise dissection of molecular mechanisms.

• Modeling Human Disease: The RANK/RANKL/OPG axis is implicated in diseases such as osteoporosis, Paget’s disease, rheumatoid arthritis, and bone metastases. Using recombinant human RANK allows for the development of human-relevant models to study disease mechanisms and screen potential therapeutics.

• Protein-Protein Interaction Assays: Recombinant RANK is used in binding studies to characterize interactions with RANKL, OPG, and other modulators, supporting drug discovery and antibody development.

• Immunological Research: RANK is involved in the regulation of T-cell and dendritic cell interactions, making recombinant RANK valuable for immunology studies, including those on immune tolerance and inflammation.

• High Purity and Consistency: Recombinant proteins offer high purity, defined activity, and batch-to-batch consistency, which are critical for reproducible results in biochemical and cell-based assays.

• Versatility in Assays: Recombinant human RANK can be used in a variety of applications, including:

  • Cell signaling and differentiation assays
  • ELISA and other immunoassays as a standard or capture reagent
  • Structural biology studies to resolve RANK-ligand complexes

• Reduction of Animal Use: Recombinant human proteins facilitate the use of human cell-based systems and organoids, reducing reliance on animal models and improving translational relevance.

In summary, recombinant human RANK is a critical reagent for elucidating the biology of bone and immune systems, modeling human disease, and supporting the development of targeted therapies.

Yes, recombinant human RANK can be used as a standard for quantification or calibration in ELISA assays, provided it is well-characterized and compatible with your assay system. Recombinant proteins are commonly used as standards in ELISA protocols to generate standard curves for quantitative analysis.

Key considerations and best practices:

• Purity and Characterization: The recombinant RANK protein should be highly purified and its concentration accurately determined, ideally by methods such as HPLC or spectrophotometry.
• Assay Compatibility: The ELISA antibodies must recognize both natural and recombinant forms of RANK. Most commercial human RANK ELISA kits are validated to detect recombinant RANK as well as endogenous protein.
• Standard Curve Preparation: Prepare serial dilutions of the recombinant RANK protein in the same buffer or matrix as your samples to minimize matrix effects and ensure accurate quantification.
• Validation: Confirm that the recombinant standard yields a linear and reproducible standard curve within the assay’s dynamic range. Recovery experiments (spiking known amounts into sample matrices) can further validate accuracy.
• Documentation: Use the manufacturer’s instructions for your ELISA kit, as some kits provide lyophilized recombinant human RANK specifically for use as a standard.

Limitations:

• Recombinant standards may differ in post-translational modifications compared to native proteins, which can affect antibody binding in rare cases. Most ELISA kits are designed to minimize this issue, but it is advisable to check the kit’s validation data.
• For clinical or diagnostic applications, additional validation may be required to ensure equivalence between recombinant and native RANK.

Summary Table: Use of Recombinant Human RANK as ELISA Standard

In conclusion, recombinant human RANK is suitable as a standard for ELISA quantification, provided it is properly validated and compatible with your assay system.

Recombinant Human RANK has been validated primarily for use in bioassays, ELISA (as a standard), cell culture, and surface plasmon resonance (SPR) in published research.

Key validated applications include:

• Bioassays: Recombinant Human RANK is widely used to study the RANK/RANKL signaling pathway, particularly in osteoclastogenesis, bone metabolism, and immune cell function. Published studies have employed it to assess cellular responses such as differentiation, activation, and cytokine production in whole cell systems.

• ELISA (Standard): It serves as a standard for quantifying RANK or RANKL in biological samples, supporting research into bone resorption, osteoporosis, and related disorders.

• Cell Culture: Recombinant RANK is used to stimulate or modulate cells in vitro, enabling functional studies of osteoclasts, macrophages, and other cell types involved in bone and immune system biology.

• Surface Plasmon Resonance (SPR): SPR assays have validated recombinant RANK for studying protein-protein interactions, such as binding kinetics with RANKL or other ligands, which is critical for drug discovery and mechanistic studies.

Representative published research applications:

• Osteoclastogenesis and bone metabolism: Studies have used recombinant RANK to investigate the molecular mechanisms of osteoclast differentiation and function, including the effects of microRNAs, cytokines, and disease states on RANK signaling.

• Cancer metastasis and bone disease: Research has validated recombinant RANK in models exploring the role of RANK/RANKL in cancer cell interactions with bone, metastatic growth, and skeletal-related events.

• Immunology: Recombinant RANK is used to study dendritic cell survival, T cell activation, and macrophage function, reflecting its broader role in immune regulation.

In summary, bioassay, ELISA standard, cell culture, and SPR are the principal validated applications for recombinant human RANK in published research, supporting studies in bone biology, cancer, and immunology.

To reconstitute and prepare Recombinant Human RANK protein for cell culture experiments, dissolve the lyophilized protein in sterile water or PBS to the recommended concentration, typically 0.1–1 mg/mL, and include a carrier protein such as 0.1% BSA if specified for stability.

Detailed protocol:

• Reconstitution:

  • Check the product datasheet for the recommended buffer and concentration. For many recombinant human RANK proteins, reconstitution is performed in sterile water or PBS at concentrations such as 1 mg/mL or 100 µg/mL.
  • If the formulation is carrier-free, use sterile PBS. If the formulation includes BSA or trehalose, reconstitute in sterile PBS containing at least 0.1% BSA for enhanced stability and to prevent adsorption to tube walls.
  • Gently add the buffer to the lyophilized protein and allow it to dissolve at room temperature for 15–30 minutes with gentle agitation. Avoid vigorous shaking or vortexing to prevent protein denaturation and foaming.

• Aliquoting and Storage:

  • Once fully dissolved, aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade protein activity.
  • For short-term use (up to one week), store aliquots at 2–8°C.
  • For long-term storage, aliquot and freeze at –20°C to –80°C. Addition of carrier proteins (e.g., BSA, FBS, or trehalose) and/or 5–50% glycerol can further stabilize the protein during freezing.

• Preparation for Cell Culture:

  • Thaw aliquots on ice and dilute to the desired working concentration in cell culture medium immediately before use.
  • If performing serum-free culture or in vivo experiments, avoid animal-derived carrier proteins and use trehalose or other non-animal stabilizers.
  • Filter sterilize the final solution if necessary, using a 0.2 µm filter, especially if the protein will be added directly to cell cultures.

• Application:

  • Add the reconstituted protein directly to cell culture media at the required concentration for your experiment. For functional assays, typical working concentrations range from 1–10 ng/mL depending on the cell type and assay conditions.

Key considerations:

• Always consult the specific product datasheet for buffer recommendations and concentration.
• Avoid repeated freeze-thaw cycles by aliquoting.
• Use carrier proteins for stability unless contraindicated by your experimental design.

This protocol ensures optimal solubility, stability, and biological activity of recombinant human RANK protein for cell culture applications.