Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL), also known as Tumor necrosis factor ligand superfamily member 11 (TNFSF11), TNF-related activation-induced cytokine (TRANCE), osteoprotegerin ligand (OPGL), and osteoclast differentiation factor (ODF) is a member of the tumor necrosis factor (TNF) ligand family. RANKL regulates bone remodeling and the development of the thymus, lymph nodes and mammary glands. Overproduction of RANKL is implicated in a variety of degenerative bone diseases, such as rheumatoid arthritis and psoriatic arthritis.
Kingfisher Biotech’s Mouse RANKL/TNFSF11 activity was accessed and shown to be active and comparable with other commercially available Human RANKL protein. This work was done by an independent lab with expertise in osteoclast cell culture. Both samples of RANKL were reconstituted with 0.1% BSA in PBS prior to assay. Kingfisher Biotech’s RANKL protein concentration was determined by BCA. Protein determination of the competitor’s protein is unknown. Kingfisher Biotech’s mouse RANKL was expressed in yeast so it is naturally endotoxin-free and post-translationally modified. Human RANKL purchased from Competitor A was produced in E. coli.
Method
RAW 264.7 cells (1.3 × 104/ml) were cultured in 24-well plates for 5 days in the presence of RANKL (100 ng/ml) and M-CSF (10 ng/ml). The culture media was changed every alternate day and replaced with fresh media and cytokines. At the end of the culture period, cells were fixed in 2% glutaraldehyde in PBS for 20 min and stained for TRAP activity (Sigma, 387A). TRAP positive multinucleated cells containing three or more nuclei were scored microscopically. The experiment was repeated 15 days after the initial experiment and yielded similar results.
Assay Results
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