Nori Mouse RANK ELISA Kit Summary

Alternative names for RANK: Receptor Activator of Nuclear Factor κ B, TRANCE Receptor, TNFRSF11A, Osteoclast differentiation factor receptor, ODFR, CD265

O35305

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 κ B (RANK), also known as TRANCE Receptor or TNFRSF11A, is member of the tumor necrosis factor receptor (TNFR) molecular sub-family. RANK is the receptor for RANK-Ligand (RANKL) and part of the RANK/RANKL/OPG (osteoprotegerin) signaling pathway that regulates osteoclast differentiation and activation. It is associated with bone remodeling and repair, immune cell function, lymph node development, thermal regulation, and mammary gland development. OPG is a decoy receptor for RANK, and regulates the stimulation of the RANK signaling pathway by competing for RANKL. The cytoplasmic domain of RANK binds TRAFs 1, 2, 3, 5, and 6 which transmit signals to downstream targets such as NF-κB and JNK. RANK is constitutively expressed in multiple cells and tissues.^[1]. Most commonly, activation of NF-κB is mediated by RANKL, but over-expression of RANK alone is sufficient to activate the NF-κB pathway.^[2] RANKL is found on the surface of stromal cells, osteoblasts, and T cells.^[3] RANKL binds to RANK, which then binds to TRAF6. TRAF6 stimulates the activation of the c-jun N-terminal kinase (JNK) and nuclear factor kappa-b (NF-kB) pathways which trigger differentiation and activation of osteoclasts. This system is balanced by the relative expression of OPG to RANKL, which are highly regulated by many factors. An overexpression of RANKL can cause an overproduction and activation of osteoclasts, which break down bone. The balance between RANKL and OPG is a target for therapy in many diseases including estrogen deficiency-associated osteoporosis, rheumatoid arthritis, Paget’s disease, periodontal disease, and bone tumors and malignancies.^[4] The destruction of bone releases more growth factors and RANKL which induces more osteoclastogenesis, triggering a viscous cycle of bone destruction that is seen in metastatic bone tumors.^[6]

References

1. Sattler AM, et al. (2004). Calcif. Tissue Int. 74 (1): 103–6.
2. Anderson DM, et al. (1997). Nature 390 (6656): 175–179.
3. Wong BR, et al. (1999). J. Leukoc. Biol. 65 (6): 715–24.
4. Hofbauer, et al. (2001). Journal of Molecular Medicine 79 (5-6): 243–253.
5. Schramek, et al. (2011). IBMS BoneKEy 8 (5): 237–256.