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Extra info for Amyloid Precursor Protein: A Practical Approach
4 DISCUSSION In this chapter, we have described assays based on the binding of a radioiodinated or unlabeled monoclonal antibody to investigate the aspects of APP trafficking in cultured cells. The use of an antibody for kinetic analysis of APP trafficking from the cell surface may be the only possible approach because of the lack of a physiological soluble ligand for APP at the cell surface. Biotinylation of cell surface APP is possible but this would be very cumbersome for obtaining kinetic information.
3. 0 M NaCl gradient in column equilibration buffer. 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.
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.