This Mouse CES5 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CES5 protein (Cat: 50620-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain of mouse CES5 (NP_766347.1) (Met 1-His 556) was expressed, with a C-terminal polyhistidine tag.
The secreted recombinant mouse CES5 comprises 541 amino acids and has a calculated molecular mass of 60.6 kDa. As a result of glycosylation, the recombinant protein migrates as an approximately 52 kDa band in SDS-PAGE under reducing conditions.
Mouse CES5 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.