Description

Nori Human RANKL ELISA Kit Summary

Alternative names for RANKL: Receptor activator of nuclear factor kappa-B ligand (RANKL), TNFSF11

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background:

Receptor activator of nuclear factor kappa-B ligand (RANKL) is a protein that is encoded by the TNFSF11 gene.^[1] RANKL is known as a type II membrane protein and is a member of the tumor necrosis factor (TNF) superfamily.^[2] RANKL has been identified to affect the immune system and control bone regeneration and remodeling. RANKL may be used as an apoptosis regulator gene, a binding partner of OPG, a ligand for the receptor RANK and control proliferation by modifying protein levels of Id4, Id2 and cyclin D1.^[3] RANKL is expressed in several tissues and organs and high protein expression of RANKL are commonly detected in the lung, thymus and lymph nodes.^[3] The level of RANKL expression does not linearly correlate to the effect of this ligand., While bone marrow expresses low levels of RANKL, it plays a critical role for adequate bone metabolism, this surface-bound molecule (also known as CD254) found on osteoblasts serves to activate osteoclasts, which are critically involved in bone resorption. Osteoclastic activity is triggered via the osteoblasts’ surface-bound RANKL activating the osteoclasts’ surface-bound receptor activator of nuclear factor kappa-B (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. T cell activation was reported to induce expression of this gene and lead to an increase of osteoclastogenesis and bone loss. This protein was shown to activate antiapoptotic kinase AKT/PKB through a signaling complex involving SRC kinase and tumor necrosis factor receptor-associated factor 6 (TRAF6), which indicated this protein may have a role in the regulation of cell apoptosis.

References

1. Anderson DM, et al. (1997). Nature 390 (6656): 175–9.
2. Hanada R, et al. (2011). J. Mol. Med. 89 (7): 647–56.
3. Wada T, et al. (2006). Trends Mol Med 12 (1): 17–25.

Product Citation