Description

Nori Mouse 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

40.        Zucca E et al. (2016) Evaluation of amniotic mesenchymal cell derivatives on cytokine production in equine
alveolar macrophages: an in vitro approach to lung inflammation. Stem Cell Research and Therapy 7:137.   Article
Product used and cited: Nori Equine TNFα/IL-6/TGFβ1 ELISA kits. Impact factor: 4.731.

91.        Witonsky S et al. (2019) Can levamisole upregulate the equine cell-mediated macrophage (M1) dendritic
cell (DC1) T-helper 1 (CD4 Th1) T-cytotoxic (CD8) immune response in vitro? J Vet Internal Med 33(2):889-96.
Impact factor: 2.286.
Products used and cited: Nori Equine IL-6 ELISA Kit.

37.        Siemieniuch MJ et al. (2016) Type of inflammation differentially affects expression of interleukin 1β and 6, tumor
necrosis factor-α and toll-like receptors in subclinical endometritis in mares. PLOS ONE 11(5):e0154934. Doi:10.137
/journal.pone.0154934. Impact factor: 2.806.
Product used and cited: Nori Equine IL-6 ELISA kit.

48. Water EVD et al. (2016) The preventive effects of two nutraceuticals on experimentally induced acute synovitis. Equine Veterinary Journal DOI:10.1111/evj.12629. Impact factor: 2.40.

Product used and cited: Nori Equine IL-6 ELISA Kit GR106001, GR106088.

81.        Abass M et al. (2018) Local mepivacaine before castration of horses under medetomidine
isoflurane balanced anesthesia is effective to reduce perioperative nociception and cytokine release.
Equine Veterinary Journal, 50:733-738. Impact factor: 2.022
Produce used and cited: Nori Equine IL-6 and TNF-alpha ELISA Kit.

96.        Broeckx SY et al. (2019) The use of equine chondrogenic-induced mesenchymal stem cells as a treatment for osteoarthritis:
A randomized, double-blinded, placebo-controlled proof-of-concept study. Equine Vet J 51(6): 787-94. Impact factor: 2.022.
Products used and cited: Nori Equine IL-6 and TGFβ3 ELISA Kits.

110.        Hussein H et al. (2016) Cathepsin K inhibition renders equine bone marrow nucleated cells hypo-responsive
to LPS and unmethylated CpG stimulation in vitro. Comparative Immunology, Microbiology and Infectious Diseases
45: 40-7. Impact factor: 2.05.
Products used and cited: Nori Equine IL-1β, IL-6 and TNFα ELISA Kits.

126. Broeckx SY et al. (2019) Evaluation of an osteochondral fragment-groove procedure for induction of
metacarpophalangeal joint osteoarthritis in horses. Am J Vet Res 80(3):246-58. Impact factor: 1.07.
         Products used and cited: Nori Equine IL-6 and TGFb3 ELISA Kits (GR106001, GR106134).