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: 使用指南
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 can mediate IL-6 signals into the cells through the interaction of gp130. The resulting IL-6/sIL-6R protein complex is also capable of binding to gp130 and inducing intracellular signaling. Through this so-called 'trans-signaling' mechanism, IL-6 can 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.
Barill S, et al. (2000) The role of interleukin-6 and interleukin-6/interleukin-6 receptor-alpha complex in the pathogenesis of multiple myeloma. Eur Cytokine Netw. 11(4): 546-51.
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.