By Weiming Xia, Huaxi Xu
Within the look for a good remedy for Alzheimer's illness, APP is a special version protein that illustrates the wide variety of uncomplicated and complicated characterization recommendations to be had. Exploring a number of organic innovations to elucidate the constitution and serve as of this transmembrane protein, this article offers every one approach with precise, step by step protocols to accomplish reproducible effects and supply a framework for learning different membrane proteins.
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Extra info for Amyloid Precursor Protein: A Practical Approach
Under these circumstances, the medium and cell lysate should first be precipitated with trichloroacetic acid to recover the antibody-bound radioactivity only. As a consequence of the precipitation, the total radioactivity will be less than 100%. 4 STEADY STATE LEVEL OF APP ENDOCYTOSIS To measure the rate of APP endocytosis under steady state conditions, the radiolabeled antibody is allowed to bind at 37˚C, resulting in a concomitant uptake of radioiodinated antibody by APP internalization. fm Page 24 Wednesday, November 3, 2004 2:59 PM the internalization of transferrin receptor under steady state conditions.
Cells are spread onto MD plates and grown at 30oC for 2 to 5 days. 12. Colonies are grown in 5 ml YPD for 2 days, centrifuged and then grown in 1 ml BMMY for 2 days. 13. Expressing clones are identified by silver stain sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the culture supernatants. The expressing clones are then grown in culture to produce large amounts of GFD for crystallography and biological assays as described below. 3 METHOD FOR EXPRESSION 1. Shaker flask cultures are grown by inoculating a suitable high-level expression clone into 500 ml YPD in a 2-liter baffled flask.
Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated α-secretase cleavage of the Alzheimer amyloid protein precursor. J. Biol. Chem. 273, 27765–27767, 1998. fm Page 17 Wednesday, November 3, 2004 2:59 PM 30. S. et al. Cleavage of amyloid β-peptide during constitutive processing of its precursor. Science 248, 1122–1124, 1990. 31. L. Evidence that β-amyloid protein in Alzheimer’s disease is not derived by normal processing. Science 248, 492–495, 1990. 32. J. β-Amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms.
Amyloid Precursor Protein: A Practical Approach by Weiming Xia, Huaxi Xu