Free soluble RANKL ELISA Assay Kit

$870.00

The Eagle Biosciences Free soluble RANKL ELISA Assay Kit is an enzyme immunoassay for the quantitative determination of free, soluble, uncomplexed human RANKL in serum or heparin plasma. The Free soluble RANKL ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: BI-20462 Categories: , ,

Free soluble RANKL ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: (0 pmol/l + 3 SD): 0.01 pmol/l / 0.008pmol/l
Dynamic Range: 0 to 2 pmol/l
Incubation Time: 3.5 hours
Sample Type: Human serum, Heparin Plasma
Sample Size: 150 µL
Controls Included

Product Developed and Manufactured by Biomedica


Unit conversion: 1 pg/ml = 0.05 pmol/l (MW: 20 kD, monomer)

Additional Information

Assay Background

RANKL, the receptor activator of nuclear factor kappa B ligand, a member of the tumor necrosis factor (TNF) family (https://www.uniprot.org/uniprot/O14788), is the main stimulatory factor for the formation of mature osteoclasts and is essential for their survival. RANKL activates its specific receptor RANK, located on osteoclasts and dendritic cells. The effects are counteracted by OPG which acts as an endogenous soluble receptor antagonist (see: BI-20403 – OPG ELISA). The major source of RANKL are osteocytes, former osteoblasts that become embedded within the mineralized bone matrix. RANKL is a ~35 kD type II transmembrane-type protein and is cleaved to release a soluble biologically active product that forms a homotrimer.

RANKL and its specific receptor RANK are not only key regulators of bone remodeling but also play an essential role in immunobiology, e.g. lymph node formation, establishment of the thymic microenviroment, mammary gland development during pregnancy, bone metastasis in cancer and sex-hormone, progestin-driven breast cancer, thermoregulation, and finally in the development of type 2 diabetes mellitus.

Assay Procedure

  1. Prewash wells with 300 µl diluted WASHBUF (wash buffer, natural cap) five times. Remove remaining WASHBUF by tapping plate against paper towel after the last wash.
  2. Add 50 µl ASYBUF (assay buffer, red cap) into each well. Pipette additional 150 µl ASYBUF into well marked as blank.
  3. Pipette 150 µl STD/SAMPLE/CTRL (Standard/Sample/Control) in duplicate into respective wells, except blank.
  4. Cover tightly and incubate at room temperature (18-24°C) for 2 hours.
  5. Aspirate and wash wells with 300 µl diluted WASHBUF (wash buffer, natural cap) five times. Remove remaining WASHBUF by tapping plate against paper towel after the last wash.
  6. Add 200 µl AB (biotinylated anti sRANKL antibody, green cap) into each well, except blank. Swirl gently.
  7. Pipette additional 200 µl ASYBUF (assay buffer, red cap) into well marked as blank.
  8. Cover tightly and incubate at 4°C (2-8°C) over night (18-24 hours).
  9. Aspirate and wash wells with 300 µl diluted WASHBUF (wash buffer, natural cap) fives times. Remove remaining WASHBUF by tapping plate against paper towel after the last wash.
  10. Add 200 µl CONJ (conjugate, amber cap) into each well.
  11. Cover tightly and incubate at room temperature (18-24°C) for 1 hour in the dark.
  12. Aspirate and wash wells with 300 µl diluted WASHBUF (wash buffer, natural cap) fives times. Remove remaining WASHBUF by tapping plate against paper towel after the last wash.
  13. Add 200 µl SUB (substrate, blue cap) into each well.
  14. Incubate at room temperature (18-24°C) for 30 min in the dark.
  15. Add 50 µl STOP (stop solution, white cap) into each well.
  16. Measure absorbance immediately at 450 nm with reference 630 nm if available.

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Typical Standard Curve

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Manual

Product Manual


Publications

Citations

  • Immunology and bone. Danks L and Takayanagi H, J Biochem 2013; 154: 29-39.
    Physiology and pathophysiology of the RANKL/RANK system. Hanada R et al., Biol Chem 2010; 391(12): 1365-1370.
  • Evidence for osteocyte regulation of bone homeostasis through RANKL expression. Nakashima T et al., Nat Med 2011; 17(10): 1231-1234.
  • RANKL Employs Distinct Binding Modes to Engage RANK and the Osteoprotegerin Decoy Receptor. Nelson CA et al., Structure, Nov 2012; 20(11): 1971-1982.
  • Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer. Schramek D et al., Nature 2010; 468(7320): 98-102.
  • RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis. Gonzalez-Suarez E et al., Nature 2010; 468: 103-107.
  • Central regulation of body temperature by RANKL/RANK pathway. Hanada R and Penninger JM, Clin Calcium 2011; 21(8): 1201-1208.
  • Blockade of receptor activator of nuclear factor-κB (RANKL) signaling improves hepatic insulin resistance and prevents the development of diabetes mellitus. Kiechl S et al., Nature Medicine 2013; 19(3):358-363.