This Mouse CST6 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CST6 protein (Cat: 50916-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain of mouse CST6 (NP_082899.1) (Met 1-Ala 152) was expressed, with a C-terminal polyhistidine tag.
The secreted recombinant mouse CST6 comprises 132 amino acids and has a calculated molecular mass of 15.2 kDa. As a result of glycosylation, the apparent molecular mass of rmCST6 is approximately 20 kDa in SDS-PAGE under reducing conditions.
Mouse CST6 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.
Cystatin E/M, also referred to as CST6, is a member of type 2 cysteine proteinase inhibitors of the cystatin superfamily, and inhibits papain and cathepsin B. Cystatin E is a low molecular mass secreted protein existing in both a glycosylated (17 kDa) and an unglycosylated (14 kDa) form, with two characteristic intrachain disulfide bridges. Expression of cystatin M/E is found to be restricted to the epidermis, more specifically in the stratum granulosum, sweat glands, sebaceous glands, and the hair follicles. In addition to its function as a cysteine protease inhibitor, cystatin M/E also serves as a target for cross-linking by transglutaminases. Accordingly, cystatin M/E was suggested to be involved in barrier formation and maintenance. Furthermore, studies have revealed that cystatin M/E is frequently epigenetically inactivated during breast carcinogenesis, and thus be regarded as a candidate of tumour suppressor gene.
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