Human CST6 HEK293 Overexpression Lysate

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Human CST6 HEK293 Overexpression Lysate: Product Information

Product Description
This Human CST6 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CST6 protein (Cat: 10438-H08H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Species
Human
Sequence Information
A DNA sequence encoding the human cystatin E/M protein (NP_001314.1) (Met 1-Met 149) was expressed with a C-terminal polyhistidine-tag.
Molecule Mass
The mature form of recombinant human cystatin E/M consists of 132 amino acids and predicts a molecular mass of 15 kDa. It exists as two major forms with an identical N-terminal sequence. As a result of glycosylation, rh cystatin E/M migrates as doublets with the molecular mass of approximately 15 and 21 kDa respectively in SDS-PAGE under reducing conditions.

Human CST6 HEK293 Overexpression Lysate: Usage Guide

Preparation Method
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.
Lysis Buffer
Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.
Recommend Usage
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.
Sample Buffer
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.
Application
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.

CST6 Background Information

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.
Full Name
cystatin E/M
References
  • Ritonja A., et al.,(1985), Amino acid sequence of the intracellular cysteine proteinase inhibitor cystatin B from human liver. Biochem. Biophys. Res. Commun. 131:1187-1192.
  • Pennacchio L.A., et al., (1996), Mutations in the gene encoding cystatin B in progressive myoclonus epilepsy (EPM1).Science 271:1731-1734.
  • Ghaemmaghami S., et al.,(2003), Global analysis of protein expression in yeast.Nature 425:737-741.
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