Human Cystatin D HEK293 Overexpression Lysate: Product Information
This Human Cystatin D overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Cystatin D protein (Cat: 10506-H08H) from the overexpression lysate was verified.
A DNA sequence encoding the pro form of human Cystatin D (NP_001891.2) (Met 1-Val 142) with a C-terminal polyhistidine tag was expressed.
The secreted mature recombinant human CST5 comprises 133 amino acids with the predicted molecular mass of 15.2 kDa as estimated by SDS-PAGE under reducing conditions.
Human Cystatin D HEK293 Overexpression Lysate: Usage Guide
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.
1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube.
2. Re-dissolve the pellet using 200μL pure water and boil for 2-5 min.
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Stability & Storage
Store at 4℃ for up to twelve months from date of receipt. After re-dissolution, aliquot and store at -80℃ for up to twelve months. Avoid repeated freeze-thaw cycles.
Western Blot (WB) Optimal dilutions/concentrations should be determined by the end user.
Human Cystatin D HEK293 Overexpression Lysate: Alternative Names
Human CST5 Overexpression Lysate; Human MGC71922 Overexpression Lysate
Cystatin D Background Information
Cystatins are natural inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases. The mammalian cystatin superfamily members are of three major types, including the type 1 cystatins (stefins), type 2 cystatins and the kininogens. As a member of type 2 cystatin, cystatin D is a single-domain protein and also has cysteine residues that form disulfide bridges. In contrast with the wider distribution of all the other family 2 cystatins, cystatin D is tissue-restricted expressed and has been found only in saliva and tears. and meanwhile, it displays an inhibition profile with a preferential inhibition on cathepsin S, H, L. Although the exact functions are largely unknown, it has reported that cystatin D is involved in the inhibition of virus replication and apoptosis.
Freije J.P., et al.,(1991), Structure and expression of the gene encoding cystatin D, a novel human cysteine proteinase inhibitor. J. Biol. Chem. 266:20538-20543.
Freije J.P., et al., (1993), Human cystatin D. cDNA cloning, characterization of the Escherichia coli expressed inhibitor, and identification of the native protein in saliva.J. Biol. Chem. 268:15737-15744.
Deloukas P., et al.,(2001), The DNA sequence and comparative analysis of human chromosome 20.Nature 414:865-871.
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