期刊
PROTEOMICS
卷 12, 期 7, 页码 932-937出版社
WILEY
DOI: 10.1002/pmic.201100639
关键词
Peptide microarray; Phosphorylation; Phos-tag Biotin; QCM; Technology; Western blotting
资金
- Japan Society for the Promotion of Science (JSPS) [23117522, 22-3287]
- Ministry of Education Culture, Sports, Science, and Technology (MEXT) [22790037]
- [22390006]
- [22590037]
- [19107002]
- Grants-in-Aid for Scientific Research [22590037, 22790037, 22390006] Funding Source: KAKEN
We have previously shown that the dinuclear zinc(II) complex Phos-tag and its derivatives act as phosphate-capture molecules in aqueous solution under conditions of neutral pH. In this study, our aim was to develop more-advanced applications for the detection of phosphopeptides and phosphoproteins by using several newly synthesized Phos-tag derivatives, including a bisbiotinylated Phos-tag (BTL-108), a tetrakisbiotinylated Phos-tag (BTL-109), and a monobiotinylated Phos-tag with a dodeca(ethylene glycol) spacer (BTL-111), as well as the commercially available product BTL-104. Among these complexes, BTL-111 showed the best performance in Western blotting by an ECL system using HRP conjugated streptavidin. In addition, in a quartz-crystal microbalance analysis of a phosphoprotein, the presence of the long hydrophilic dodeca(ethylene glycol) spacer in a novel Phos-tag sensor chip coated with BTL-111 resulted in a greater sensitivity than was achieved with a similar chip coated with BTL-104. Moreover, a peptide microarray technique using the ECL system and BTL-111 permitted high-throughput assays for the specific and highly sensitive detection of protein kinase activities in cell lysates.
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