By Weiming Xia, Huaxi Xu
Within the look for a good therapy for Alzheimer's ailment, APP is a different version protein that illustrates the big range of easy and complicated characterization recommendations on hand. Exploring numerous organic suggestions to explain the constitution and serve as of this transmembrane protein, this article provides each one strategy with distinctive, step by step protocols to accomplish reproducible effects and supply a framework for learning different membrane proteins.
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Incubate the mixture for 6 min, gently flicking the tube every 30 sec. During this incubation, iodous ions undergo electrophilic attack at the ortho ring positions of tyrosine residues. 6. Remove the labeling solution and add it to a pre-equilibrated NAP-5 column. Drain the column and discard the flow-through. 7. Elute the column with 980 µl DPBS. Add 20 µl of 100 mg/ml crystalline BSA as a carrier protein to a final concentration of 2 mg/ml. 8. Determine radiospecific activity of the labeled antibody in a gamma counter.
On the following day, inoculate the overnight culture into 500 ml YPD in a 2-liter flask. 5. 6. Centrifuge (1500 × g for 5 min) and resuspend cells in 500 ml ice cold water. 7. Centrifuge and resuspend in 250 ml ice-cold water. 8. Centrifuge and resuspend in 20 ml ice-cold 1 M sorbitol. 9. Centrifuge and resuspend in 1 ml ice-cold 1 M sorbitol. 10. 2-cm electroporation cuvette on ice. Electroporate according to manufacturer’s recommended conditions for yeast. 11. Cells are spread onto MD plates and grown at 30oC for 2 to 5 days.
After 30 min, the secreted pool remains stable whereas the internalized fraction declines, presumably because of degradation. If the experiment is prolonged to more than 30 min, one should take into account that some of the radiolabeled antibody may be degraded and therefore free radioactivity-liberated. 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%.
Amyloid Precursor Protein A Practical Approach by Weiming Xia, Huaxi Xu