Mouse RANKL protein, His and GST tag

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 protein that in humans is encoded by the TNFSF11 gene. RANKL is a member of the tumor necrosis factor (TNF) cytokine family, it binds to RANK on cells of the myeloid lineage and functions as a key factor for osteoclast differentiation and activation. It may also bind to osteoprotegerin, a protein secreted mainly by cells of the osteoblast lineage which is a potent inhibitor of osteoclast formation by preventing binding of RANKL to RANK. RANKL also has a function in the immune system, where it is expressed by T helper cells and is thought to be involved in dendritic cell maturation. This protein was shown to be a dendritic cell survival factor and is involved in the regulation of T cell-dependent immune response. Besides, Peroxisome Proliferator Activated Receptor Gamma (PPARg) has been identified as an interactor of RANKL, thus a binding ELISA assay was conducted to detect the interaction of recombinant mouse RANKL and recombinant mouse PPARg. Briefly, RANKL were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to PPARg-coated microtiter wells and incubated for 2h at 37ºC. Wells were washed with PBST and incubated for 1h with anti-RANKL pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37ºC. Finally, add 50 μl stop solution to the wells and read at 450nm immediately. The binding activity of RANKLand PPARg was in a dose dependent manner.