Fractions showing binding activity according to the various protocols were dialyzed and loaded onto SGI-1776 SulfoLink column (Pierce, Rockford, IL) with 2.4 mM 21 mer peptide (Table 1). The column was incubated with 3.4 mM 16 mer (Table 1) for 17 h. The eluates were concentrated, separated on 4–12% Tris–glycine–acrylamide and blotted for sequence analysis. Flow through fractions as well as fractions from the NaCl elution of the affinity column were processed in parallel to control for nonspecific bands.
Results
Table 2.
Comparison of the neutron three methods used for the purification of APP-binding proteins
ProtocolSourceMain purification stepsIdentified proteins
170 g pig brain1% Triton extraction, sAPPα column eluted with 17 mer for 1 hProteins of 40, 52, 56, and 220 kDa
20.6 g human brain3% Cholate extraction, 22 mer Sulfolink column eluted with 10 μM 16 mer for 1 hSerum albumin, 41 kDa novel protein
3120 g human brain0.64% Igepal extraction, 21 mer Sulfolink column eluted with 3.4 mM 16 mer for 17 hhCRMP2, Actin, 63 kDa novel protein
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LY 317615 We previously reported that cultured cerebellar
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