Mouse Cyclin E Baculovirus-Insect cells Overexpression Lysate: 产品信息
This Mouse Cyclin E overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of Cyclin E protein (Cat: 50896-M20B) from the overexpression lysate was verified.
A DNA sequence encoding the mouse CCNE1 (AAH62152.1) (Met1-Glu408) was expressed with the N-terminal polyhistidine-tagged GST tag at the N-terminus.
The recombinant mouse CCNE1/GST chimera consists of 645 amino acids and has a calculated molecular mass of 74.8 kDa. The recombinant protein migrates as an approximately 75 kDa band in SDS-PAGE under reducing conditions.
Mouse Cyclin E Baculovirus-Insect cells 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.
Cyclin E1 is a member of the highly conserved cyclin family and belongs to the E-type cyclin that functions as a regulator of S phase entry and progression in mammalian cells. Cyclin E1 serves as regulatory subunits that bind, activate, and provide substrate for its associated cyclin-dependent kinase2 (CDK2), whose activity is essential for cell cycle G1 / S transition. Over expression of this encoding gene has been found in many tumors, which results in chromosome instability and by extension, induce tumorigenesis. This protein was also found to associate with, and be involved in, the phosphorylation of NPAT protein (nuclear protein mapped to the ATM locus), which participates in cell-cycle regulated histone gene expression and plays a critical role in promoting cell-cycle progression in the absence of pRB. In general, cyclin E1, as an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression and is frequently overexpressed in cancer cells.
Honda R, et al. (2005) The structure of cyclin E1 / CDK2: implications for CDK2 activation and CDK2-independent roles. The EMBO Journal. 24: 452-63.
Geng Y, et al. (2007) Kinase-Independent Function of Cyclin E. 25(1): 127-39.