This Mouse PCSK9 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of PCSK9 protein (Cat: 50251-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the full length of mouse PCSK9 (NP_705793.1) precursor (Met 1-Gln 694) was expressed, with a C-terminal polyhistidine tag.
The secreted recombinant mouse PCSK9 consists of 671 amino acids and has a calculated molecular mass of 72.6 kDa. As a result of proteolysis and glycosylation, the recombinant protein migrates as doublet with apparent molecular mass of approximately 19 & 65 kDa, cprresponding to the prodomain and the mature form of PCSK9 respectively in SDS-PAGE under reducing conditions.
Mouse PCSK9 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.
Proprotein convertase subtilisin/kexin type 9 (PCSK9), also known as NARC1 (neural apoptosis regulated convertase), which is a newly identified human secretory subtilase belonging to the proteinase K subfamily of the secretory subtilase family. PCSK9 protein is an enzyme which in humans is encoded by the PCSK9 gene with orthologs found across many species. It is expressed in neuroepithelioma, colon carcinoma, hepatic and pancreatic cell lines, and in Schwann cells. PCSK9 protein is highly expressed in the liver and regulates low density lipoprotein receptor (LDLR) protein levels. Inhibition of PCSK9 protein function is currently being explored as a means of lowering cholesterol levels. Thereby, PCSK9 protein is regarded as a new strategy to treat hypercholesterolemia. PCSK9 protein contributes to cholesterol homeostasis and may have a role in the differentiation of cortical neurons.