通讯作者：Jia, LY (reprint author), Dalian Univ Technol, Coll Life Sci & Biotechnol, 2 Linggong Rd, Dalian 116024, Peoples R China.
合写作者：Wang, Conggang,Chen, Linli,Ren, Jun,Xie, Jian,Jia, Lingyun
发表刊物：JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
关键字：A beta; Oligomer; Comb polymer; Interaction protein; Alzheimer's disease
摘要：A detailed study of individual A beta-interacting proteins has always been a difficult task because A beta has a wide range of molecular weights and can easily form aggregates. In this study, we established a novel method for isolating A beta-interacting proteins by utilizing regular comb polymer immobilized on Sepharose CL-4B. To achieve site-directed ligation of A beta, a cysteine residue was added at the N-terminus of A beta. Asp and Asp(12), 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 Asp(12) were immobilized on Sepharose CL-4B. Next, modified A beta molecules were coupled to the carboxyl groups of Asp and Asp(12) 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 A beta was achieved. The prepared A beta-adsorbents were used to isolate A beta-interacting proteins from mice brain extracts. The results showed that the adsorption capacity of the A beta-adsorbents for proteins in mice brain extracts increased with the ages of the animals. SDS-PAGE analysis of the A beta-interacting proteins showed that many kinds of brain proteins were selectively adsorbed by the A beta adsorbents, and the levels of some of these proteins varied with the ages of the animals. The results indicated that A beta-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. (C) 2014 Elsevier B.V. All rights reserved.