This Mouse IL-6R overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of IL-6R protein (Cat: 50280-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the mouse IL6Rα (NP_034689.2) extracellular domain (Ile 44-Glu 357) was expressed, with a C-terminal polyhistidine tag.
The recombinant mouse IL6Rα comprises 349 amino acids with a predicted molecular mass of 39 kDa. As a result of glycosylation, it migrates as an approximately 55-60 kDa band in SDS-PAGE under reducing conditions.
Mouse IL-6R HEK293 Overexpression Lysate: 用法
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.
Interleukin 6 receptor (IL-6R) also known as CD126 (Cluster of Differentiation 126) is a type I cytokine receptor. The low concentration of a soluble form of IL-6 receptor (sIL-6R) acts as an agonist of IL-6 activity. In the IL-6R/CD126/IL6R system, both a membrane-bound IL-6R and a sIL-6R protein are able to mediate IL-6 signals into the cells through the interaction of gp13. The resulting IL-6/sIL-6R protein complex is also capable of binding to gp13 and inducing intracellular signalling. Through this so-called 'trans-signalling' mechanism, IL-6 is able to stimulate cells that lack an endogenous mIL-6R. High levels of IL-6 and sIL-6R have been reported in several chronic inflammatory and autoimmune diseases as well as in cancer.
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Kang KW, et al. (2007) Novel role of IL-6/SIL-6R signaling in the expression of inducible nitric oxide synthase (iNOS) in murine B16, metastatic melanoma clone F10.9, cells. Free Radic Biol Med. 42(2): 215-27.