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Materials and methods
Materials. Heparin (average molecular weight, 6000) and ThS were obtained from Sigma Chemicals. The other commercially available materials used were of reagent grade or higher.
Chemical synthesis of repeat peptides. All single-repeat peptides of MBD (i.e., R1, R2, R3, and R4) were chemically synthesized using a solid-phase peptide synthesizer. These peptides were obtained in lyophilized form, characterized by mass spectrometry and determined to be >95.0% pure by reverse-phase HPLC.
Preparation of recombinant MBD and its mutant. The gene AGI5198 and purification of the His-tagged 3RMBD and 4RMBD of human brain tau were performed as previously reported [11], and the purities of these domains were confirmed by SDS–PAGE.
The genes encoding four two-repeat (R12, R13, R23, and R34) and two three-repeat (R123 and R234) peptides were constructed in accordance with the method of Okuyama et al. [9]. The gene expressions, isolations and purifications of these mutants were also performed in accordance with the method. The purity of each peptide was checked by SDS–PAGE. The concentration of each peptide was determined by measuring UV absorption at 280 nm (ε = 1280 [mol?1 L cm?1] for Tyr residue); that of R12 was determined by comparing the band intensity of this peptide with that of R13 on SDS–PAGE.