By Weiming Xia, Huaxi Xu
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Extra resources for Amyloid Precursor Protein: A Practical Approach
237, 6, 1996. 16. P. Cellular actions of beta-amyloid precursor protein and its soluble and fibrillogenic derivatives. Physiol. Revs. 77, 1081, 1997. 17. Kitaguchi, N. et al. Novel precursor of Alzheimer’s disease amyloid protein shows protease inhibitory activity. Nature 331, 530, 1988. 18. Ponte, P. et al. A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors. Nature 331, 525, 1988. fm Page 40 Wednesday, November 3, 2004 2:59 PM 19. H et al. A heparin-binding domain in the amyloid protein precursor of Alzheimer’s disease is involved in the regulation of neurite outgrowth.
0% (v/v) methanol. 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.
4. 12,13,25 5. 0. 6. 0. 7. The pooled fractions from the heparin column are loaded onto the QHyperD column and bound proteins eluted with a 50-ml linear gradient of NaCl (0 to 500 mM) in column equilibration buffer. 8. GFD elutes at a concentration of 50 mM NaCl. 9. 2). 10. The purified GFD is concentrated between 4 and 5 mg/ml for crystallization trials. Care must be taken to maintain the purified GFD at 4oC because the protein readily forms microcrystals if allowed to warm up. 2 GFD purity as assessed by SDS-PAGE and Coomassie blue staining.
Amyloid Precursor Protein: A Practical Approach by Weiming Xia, Huaxi Xu