Product Description
This Human Sclerostin overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Sclerostin protein (Cat: 10593-H07H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Sequence Information
A DNA sequence encoding the mature form of human SOST (NP_079513.1) (Gln 24-Tyr 213) was fused with a polyhistidine tag at the N-terminus.
Molecule Mass
The recombinant human SOST is a monomeric protein consisting of 197 amino acids and predicts a molecular mass of 22.5 kDa. As a result of glycosylation, the apparent molecular mass of rhSOST is approximately 28 kDa in SDS-PAGE under reducing conditions..
Preparation Method
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
Lysis Buffer
Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.
Recommend Usage
1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube. 2. Re-dissolve the pellet using 200μL pure water and boil for 2-5 min.
Sample Buffer
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Stability & Storage
Store at 4℃ for up to twelve months from date of receipt. After re-dissolution, aliquot and store at -80℃ for up to twelve months. Avoid repeated freeze-thaw cycles.
Application
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.
Human CDD Overexpression Lysate;Human DAND6 Overexpression Lysate;Human SOST1 Overexpression Lysate;Human VBCH Overexpression Lysate
Sclerostin, the protein product of the SOST gene, is a potent inhibitor of bone formation. Sclerostin protein is widely expressed at low levels with highest levels in bone, cartilage, kidney, liver, bone marrow and primary osteeoblasts differentiated for 21 days, and was originally identified as an important regulator of bone remodeling, homeostasis, and links bone resorption and bone apposition. Recent studies have revealed that Sclerostin protein inhibits the bone growth probably by binding to the extracellular domain of the Wnt coreceptors LRP5 and LRP6 and disrupting Wnt-induced Frizzled-LRP complex formation.
References
Bellido T. (2006) Downregulation of SOST/sclerostin by PTH: a novel mechanism of hormonal control of bone formation mediated by osteocytes. J Musculoskelet Neuronal Interact. 6(4): 358-9. van Bezooijen RL, et al. (2007) SOST expression is restricted to the great arteries during embryonic and neonatal cardiovascular development. Dev Dyn. 236(2): 606-12. 
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