Human Nicastrin HEK293 Overexpression Lysate

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Human Nicastrin HEK293 Overexpression Lysate: Product Information

Product Description
This Human Nicastrin overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Nicastrin protein (Cat: 11183-H08H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Species
Human
Sequence Information
A DNA sequence encoding the human NCSTN (NP_056146.1) extracellular domain (Met 1-Glu 669) was expressed, with a polyhistidine tag at the C-terminus.
Molecule Mass
The recombinant human NCSTN consists of 647 amino acids and predictes a molecular mass of 72.4 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rh NCSTN is approximately 110-120 kDa due to glycosylation.

Human Nicastrin 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.

Human Nicastrin HEK293 Overexpression Lysate: Alternative Names

Human APH2 Overexpression Lysate; Human ATAG1874 Overexpression Lysate; Human KIAA0253 Overexpression Lysate; Human NCSTN Overexpression Lysate; Human RP11-517F10.1 Overexpression Lysate

Nicastrin Background Information

Nicastrin (NCST, or NCT), a single-pass membrane glycoprotein that harbors a large extracellular domain, is an essential component of the gamma-secretase complex. Several lines of evidence indicate that the members of these complexes could also contribute to the control of cell death. NCT controls cell death via phosphoinositide 3-kinase/Akt and p53-dependent pathways and that this function remains independent of the activity and molecular integrity of the gamma-secretase complexes. Increasing evidences have shown that Nicastrin/NCSTN plays a crucial role in gamma-cleavage of the amyloid precursor protein (APP). The glycoprotein Nicastrin is an essential component of the gamma-secretase complex, a high molecular weight complex which also contains the presenilin proteins, Aph-1 and Pen-2. The gamma-secretase complex is not only involved in APP processing but also in the processing of an increasing number of other type I integral membrane proteins. As the largest subunit of the gamma-secretase complex, Nicastrin plays a crucial role in its activation. Inhibition of NCSTN demonstrated an altered gamma-cleavage activity, suggesting its potential implication in developing Alzheimer's disease (AD). In addition, Nicastrin can function to maintain epithelial to mesenchymal transition during breast cancer progression. Anti-nicastrin polyclonal and monoclonal antibodies were able to decrease notch1 and vimentin expression and reduced the invasive capacity of breast cancer cells in vitro.
Full Name
nicastrin
References
  • He G, et al. (2007) Degradation of nicastrin involves both proteasome and lysosome. J Neurochem. 101(4): 982-92.
  • Hayashi I, et al. (2009) Single chain variable fragment against nicastrin inhibits the gamma-secretase activity. J Biol Chem. 284(41): 27838-47.
  • Ma Z, et al. (2009) Association between promoter polymorphisms of the nicastrin gene and sporadic Alzheimer's disease in North Chinese Han population. Neurosci Lett. 458(3): 136-9.
  • Pardossi-Piquard R, et al. (2009) p53-dependent control of cell death by nicastrin: lack of requirement for presenilin-dependent gamma-secretase complex. J Neurochem. 109(1): 225-37.
  • Filipovi? A, et al. (2011) Biological and clinical implications of nicastrin expression in invasive breast cancer. Breast Cancer Res Treat. 125(1): 43-53.
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