This Mouse Cystatin F/CST7 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Cystatin F/CST7 protein (Cat: 50236-M02H) from the overexpression lysate was verified.
A DNA sequence encoding the mouse CST7 (O89098) (Met1-Gln144) was expressed, fused with the Fc region of human IgG1 at the C-terminus.
The recombinant mouse CST7 /Fc is a disulfide-linked homodimer. The reduced monomer comprises 367 amino acids and has a predicted molecular mass of 41.4 KDa. The apparent molecular mass of the protein is approximately 42 KDa in SDS-PAGE under reducing conditions due to glycosylation.
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.
The cystatin superfamily members are important natural cysteine protease inhibitors present in a wide variety of organisms and are divided into three classes. Cystatin F, also known as leukocystatin and CMAP (Cystatin-like Metastasis-Associated Protein), is a type 2 cystatin and its expression is limited to hematopoietic cells, with the highest expression levels being observed in monocytes, dendritic cells, and certain types of T-cells. Furthermore, cystatin F mRNA becomes up-regulated during dendritic cell maturation, and thus suggests a specific role of cystatin F in immune regulation. Cystatin F is produced as a dimer, an inactive cathepsin inhibitor which is activated by chemical reduction. In addition, Cystatin F and its homologues have been observed expressing in various human cancer cell lines established from malignant tumors, and thus indicates a new diagnosis and prevention approach of certain human carcinomas metastasis.
cystatin F (leukocystatin)
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Morita M., et al.,(2000), Genomic construct and mapping of the gene for CMAP (leukocystatin/cystatin F, CST7) and identification of a proximal novel gene, BSCv (C20orf3).Genomics 67:87-91.