Recombinant human RANKL protein - Bioactive cytokine

Human RANKL protein - E. coli -expressed, tag-free, carrier-free

Recombinant human RANKL is a high-quality and biologically active cytokine, validated using proprietary RANKL reporter cells. This member of the Tumor Necrosis Factor (TNF) superfamily is produced in E. coli and thoroughly purified to remove endotoxins.

Recombinant human RANKL can be used together with HEK-Blue™ RANKL cells for the screening of inhibitory molecules, such as Denosumab, a therapeutic monoclonal antibody blocking the RANKL cytokine from binding to its receptor RANK (see figures).

RANKL signaling and biological functions

Key features

• Each lot is validated using HEK-Blue™ RANKL cells
• Endotoxin ≤1 EU/µg
• 0.2 µm sterile-filtered

Applications

• Standard for RANKL detection and quantification assays
• Screening and release assays for antibodies blocking RANKL signaling
• Screening and release assays for engineered RANKL

The Receptor Activator of NF-κB Ligand (RANKL) is a member of the TNF (Tumor Necrosis Factor) superfamily and plays a pivotal role in bone remodeling and dendritic cell survival. Blocking the binding of RANKL to its receptor RANK has been shown to reduce osteoporosis, prevent skeletal-related events (SREs) from bone metastasis in cancer, or improve anti-tumor immunity.

 More details

All InvivoGen products are for internal research use only, and not for human or veterinary use.

Figures

SDS PAGE of the recombinant human (h)RANKL protein. 2 μg of hRANKL was loaded on a 12% Mini-PROTEAN® TGX StainFree™ Precast Gel (Bio-Rad). Detection was performed as per the manufacturer’s instructions. A band was detected at ~19 kDa.

Dose-response in HEK-Blue™ RANKL cells to recombinant hRANKL protein. Cells were stimulated with increasing concentrations of recombinant human (h)RANKL. After overnight incubation, the NF-κB-induced SEAP activity was determined using QUANTI-Blue™, a SEAP detection reagent. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

Dose-dependent inhibition of HEK-Blue™ RANKL cells response using Denosumab biosimilar. A serial dilution of Denosumab, a biosimilar Anti-hRANKL-hIgG2 monoclonal antibody (mAb) was incubated with 3 ng/ml of recombinant human RANKL for 2 hours prior to the addition of the HEK-Blue™ RANKL cells. After overnight incubation, the NF-kB response was determined using QUANTI-Blue™ Solution, a SEAP detection reagent. Data are presented as percentage of activity.

Specifications

Source: E. coli

Species: Human

Alternative names: TNFSF11, Tumor necrosis factor ligand superfamily member 11, TNF-related activation-induced cytokine, TRANCE

Carrier: Carrier-free

Tag: Tag-free

Accession number: O14788

Molecular weight: ~ 20 kDa (SDS-PAGE)

Solubility: 100 μg/ml in water

Formulation: Solution containing Tris-HCl and NaCl

Form: Lyophilized

Reconstitution buffer: 1X PBS with 0.1 % BSA or HSA (not provided)

Purity: ≥ 95% (SDS-PAGE)

Endotoxin: The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK‑Blue™ TLR4 cells.

Tested applications: Cellular assays

Quality control: Each lot is functionally tested and validated.

Contents

Recombinant human RANKL is provided as a lyophilized powder and is available in two quantities:

• rcyec-hrankl​: 10 μg 
• rcyec-hrankl-5: 50 μg (5 x 10 μg)

 Recombinant human RANKL is shipped at room temperature.

Upon receipt, the product should be stored at -20°C.

 Avoid repeated freeze-thaw cycles.

Details

RANKL background

Receptor Activator of NF-κB Ligand (RANKL), also known as member 11 of the tumor necrosis factor (TNF) superfamily (TNFSF11) or TNF-related activation-induced cytokine (TRANCE), exists as a transmembrane or soluble protein produced by osteoblasts and activated T cells [1]. RANKL binds to its receptor RANK via an obligate trimer configuration [1, 2]. RANKL/RANK signaling plays a pivotal role in bone remodeling and dendritic cell survival, thereby enhancing the induction of T cell responses [1]. Upon RANKL binding, RANK trimers recruit TNF receptor-associated factor (TRAF) adaptor proteins, such as TRAF6, to TRAF-binding motifs within their cytoplasmic domains [1]. The TRAF6 signaling cascade results in the activation of NF-κB and AP-1 transcription factors. Multiple efforts have focused on the development of anti‑RANKL antibodies or small‑molecule inhibitors for blocking RANKL/RANK signaling to reduce osteoporosis, prevent skeletal-related events (SREs) from bone metastasis in cancer, or improve anti-tumor immunity [1-3].

1. Cheng ML. & Fong L., 2014. Effects of RANKL-targeted therapy in immunity and cancer. Front. Oncol. 3:329.
2. Ahern E. et al., 2018. Roles of the RANKL-RANK axis in anti-tumour immunity — implications for therapy. Nat. Rev. Clin. Oncol. 15:676-93.
3. Nakai Y. et al., 2019. Efficacy of an orally active small-molecule inhibitor of RANKL in bone metastasis. Bone Res. 7:1.