Add time:08/06/2019         Source:sciencedirect.com

A detailed study of individual Aβ-interacting proteins has always been a difficult task because Aβ has a wide range of molecular weights and can easily form aggregates. In this study, we established a novel method for isolating Aβ-interacting proteins by utilizing regular comb polymer immobilized on Sepharose CL-4B. To achieve site-directed ligation of Aβ, a cysteine residue was added at the N-terminus of Aβ. Asp and Asp12, which have 2 and 13 carboxyl groups, respectively, were selected as the carriers for the regular comb polymer. Firstly, the N-termini of Asp and Asp12 were immobilized on Sepharose CL-4B. Next, modified Aβ molecules were coupled to the carboxyl groups of Asp and Asp12 using bromoethylamine as a spacer. To obtain homogeneous comb polymer, the efficiency of the reaction was controlled during the synthesis process. Thioflavin T staining indicated that homogeneous Aβ was achieved. The prepared Aβ-adsorbents were used to isolate Aβ-interacting proteins from mice brain extracts. The results showed that the adsorption capacity of the Aβ-adsorbents for proteins in mice brain extracts increased with the ages of the animals. SDS-PAGE analysis of the Aβ-interacting proteins showed that many kinds of brain proteins were selectively adsorbed by the Aβ adsorbents, and the levels of some of these proteins varied with the ages of the animals. The results indicated that Aβ-interacting proteins could be successfully obtained through the use of immobilized comb polymer. Similar method could also be used to isolate other amyloid-interacting proteins.

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