The induction of tartrate resistant acid phosphatase (TRAP) in Raw 264.7 cells was assessed. Raw 264.7 cells were treated with increasing concentrations of hRANKL for 4 days. Cells were lysed and TRAP activity of cell extracts was assessed and OD450 determined.
The purity of recombinant hRANKL was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hRANKL and staining overnight with Coomassie Blue.
With carrier: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2 containing 20 μg BSA per 1 μg hRANKL. Carrier free: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2 containing 5% sucrose.
Stable in lyophilized state at 4°C for 1 year after receipt. Sterile stock solutions reconstituted with carrier protein are stable at 4°C for 2 months and at -20°C for 6 months. Avoid repeated freeze-thaw cycles. Maintain sterility. Storage at -20°C should be in a manual defrost freezer.
>98% as determined by SDS-PAGE of 6 μg reduced (+) and non-reduced (-) recombinant hRANKL. All lots are greater than 98% pure.
Recombinant hRANKL contains no "tags" and the nonglycosylated protein has a calculated MW of 20,484. DTT-reduced and non-reduced protein migrate as 30-35 kDa polypeptides. Lower mobility and heterogeneity in SDS-PAGE are due to glycosylation. The expected amino-terminal GSQHI of recombinant hRANKL was verified by amino acid sequencing.
The bioactivity of hRANKL was determined by measuring the ability of hRANKL to induce TRAP activity in Raw 264.7 cells. The ED50 of each lot is between 1.5-5 ng/ml.
Less than 0.01 ng endotoxin/1 μg hRANKL.
Recombinant human RANKL (hRANKL) Gly63-Asp244 (Accession #NP_143026) was expressed in human 293 cells at Cell Signaling Technology.
RANKL, also known as TRANCE or OPGL, is a member of the TNF superfamily of ligands. T cells, mammary epithelial cells, and endothelial cells can produce RANKL (1). RANKL is expressed as a type II transmembrane protein or cleaved into a soluble form by extracellular proteases, such as TACE, ADAM10, and matrix metalloproteases (1). Alternative splicing also results in the production of soluble RANKL (1). RANKL signaling is antagonized by osteoprotegerin, which functions as a soluble decoy receptor (2). RANKL plays key roles in mammary gland development and dendritic cell survival and is required for osteoclast differentiation and survival (3-6). Research studies have shown that RANKL deficiencies in both mice and humans are associated with abnormally increased bone density and defects in lymphoid organogenesis (5,6).
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