Amyloid Precursor Protein A Practical Approach by Weiming Xia, Huaxi Xu

By Weiming Xia, Huaxi Xu

Within the look for an efficient remedy for Alzheimer's illness, APP is a distinct version protein that illustrates the wide selection of simple and complicated characterization ideas on hand. Exploring quite a few organic innovations to elucidate the constitution and serve as of this transmembrane protein, this article provides every one strategy with specific, step by step protocols to accomplish reproducible effects and supply a framework for learning different membrane proteins.

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J. Biol. Chem. 268, 26571, 1993. 26. , Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press, New York, 1999. 27. M. et al. Amino-terminal region of the beta-amyloid precursor protein activates mitogen-activated protein kinase. Neurosci. Lett. 198, 52, 1995. 28. Iwasaki, W. et al. Solution structure of midkine, a new heparin-binding growth factor. EMBO J. 16, 6936, 1997. 29. Zhou, H. et al. The solution structure of the N-terminal domain of hepatocyte growth factor reveals a potential heparin-binding site.

Fluorescence intensity over a 5-min period was monitored with excitation at 328 nm and emission at 393 nm, using a detector voltage of 700 V on an Aminco Bowman luminescence spectrometer. Fit of the linear portion of the signal to a linear model produces a typical signal of 10-3 fluorescence units per second (FU/sec). 1. 15 However, this correction is not necessary in the determination of the inhibition constant Kiapp (see below) because relative initial velocities are determined at a fixed substrate concentration.

Fm Page 23 Wednesday, November 3, 2004 2:59 PM 1. Chill the cells on ice to stop membrane trafficking. 2. 2% bovine serum albumin. 3. 4 µg 125I-IG7 antibody in 400 µl binding medium per well (~7 nM final) and allow binding of the antibody for 1 hr on ice. 19 Therefore, this concentration is at least fivefold the concentration needed for half-maximal saturation. 4. Wash the cells twice with binding medium and twice with ice cold DPBS. 5. Place the cells in 2 ml of prewarmed medium and incubate at 37˚C for various periods.

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