By Weiming Xia, Huaxi Xu
Within the look for an efficient remedy for Alzheimer's illness, APP is a different version protein that illustrates the big range of uncomplicated and complicated characterization concepts on hand.
Exploring quite a few organic options to explain the constitution and serve as of this transmembrane protein, this article provides each one procedure 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
3. 0. 4. 34% (yeast nitrogen base without amino acids, 4 × 10-5% w/v) biotin, 1% dextrose, and 15 g agar for 1 liter. 5. Yeast extract peptone methanol (YPM) consisting of 1% (w/v) bacto yeast extract, 2% (w/v) peptone, and 3% (v/v) methanol. 2 METHOD FOR CLONING 1. The DNA encoding GFD is generated by polymerase chain reactin (PCR) using primers GGTCGACAAAAGAGAGGCTCTGCTGGCTGAACCCCAGATTG and GAATTCTTATACAAACTCACCAACTAAG. 2. The PCR product is cloned as a Xho1-EcoR1 fragment into the P. pastoris vector pIC9 (Invitrogen).
2. The cells are chilled on ice for 15 min and washed with ice cold DPBS to stop membrane trafficking. 3. Coverslips are then incubated with anti-APP monoclonal antibodies (1G7 alone or combined with 5A3) in cold DPBS for 1 hr on ice. 4. The cells are washed 5 times with cold DPBS and then incubated with prewarmed regular medium for various times (0 to 60 min) at 37°C. The cells are then fixed with cold 4% formaldehyde (freshly prepared from paraformaldehyde) in PBS for 15 min. 5. 3% Triton X-100 in PBS, washed 3 times with PBS, and incubated with fluorescein isothionate (FITC)-conjugated anti-mouse secondary antibody for 1 hr at room temperature.
Et al. The solution structure of the N-terminal domain of hepatocyte growth factor reveals a potential heparin-binding site. Structure 6, 109, 1998. 30. J. et al. Solution structure of the heparin-binding domain of vascular endothelial growth factor. Structure 6, 637, 1998. 31. U. et al. From differentiation to proliferation: the secretory amyloid precursor protein as a local mediator of growth in thyroid epithelial cells. Proc. Natl. Acad. Sci. USA 95, 1770, 1998. 32. A. C. Amyloid precursor protein modulates the interaction of nerve growth factor with p75 receptor and potentiates its activation of trkA phosphorylation.
Amyloid precursor protein : a practical approach by Weiming Xia, Huaxi Xu