國家衛生研究院 NHRI:Item 3990099045/1227
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    题名: Enzyme-linked immunosorbent assay for the determination of p21-activated kinase activity
    作者: Yu, JS;Chang, SH;Chan, WH;Chen, HC
    贡献者: Division of Biotechnology and Pharmaceutical Research
    摘要: An enzyme-linked immunosorbent assay (ELISA) for the measurement of p21-activated kinase (PAK) activity is described. The development of this method takes advantage of the fact that a phospho-epitope specific antibody against the regulatory autophosphorylation site sequence of PAK was successfully produced, and after being phosphorylated by PAK, a cross-linked peptide containing the autophosphorylation site of PAK could be recognized on immunoblot by this antibody. This procedure involves coating the crosslinked peptide on microtiter plates, phosphorylating the cross-linked peptide by adding active PAK plus ATP . Mg2+, and detecting peptide phosphorylation using the phosphoepitope- specific antibody and secondary antibody conjugated with alkaline phosphatase followed by reaction with p-nitrophenyl phosphate (for colorimetric detection) or fluorescein diphosphate (for fluorimetric detection). The PAK activity detected by this method was linearly proportional to the amount of kinase used in the reaction and to the duration of the kinase reaction. Furthermore, fluorimetric detection proved more sensitive than colorimetric detection in terms of both detection limit and signal magnitude. Kinase inhibitor assay revealed that the IC50 value of staurosporine obtained by this ELISA was very close to that obtained in radioassay. Besides staurosporine, the inhibitory activity of several kinase inhibitors was also tested by the PAK ELISA. The results taken together demonstrate the feasibility and efficacy of this solid phase method for the measurement of PAK activity in a non-radioactive way. Development of this method can be helpful in further high-throughput screening of potential inhibitors of this kinase.
    关键词: Biochemistry & Molecular Biology
    日期: 2001-02
    關聯: Journal of Biochemistry. 2001 Feb;129(2):243-251.
    Link to: http://wwwsoc.nii.ac.jp/jbiochem/jb/129-2/2ezajjtx.htm
    JIF/Ranking 2023: http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=NHRI&SrcApp=NHRI_IR&KeyISSN=0021-924X&DestApp=IC2JCR
    Cited Times(WOS): https://www.webofscience.com/wos/woscc/full-record/WOS:000166865000010
    Cited Times(Scopus): http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=0035113682
    显示于类别:[陳華鍵(1997-2001)] 期刊論文

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