A fluorescence resonance energy transfer-based assay to study SUMO modification in solution

被引：27

作者：

Bossis, G ^{[1
]}

Chmielarska, K

Gärtner, U

Pichler, A

Stieger, E

Melchior, F

机构：

[1] Max Planck Inst Biochem, D-82131 Martinsried, Germany

[2] Univ Gottingen, Dept Biochem 1, D-37073 Gottingen, Germany

来源：

UBIQUITIN AND PROTEIN DEGRADATION, PART A | 2005年 / 398卷

关键词：

D O I：

10.1016/S0076-6879(05)98003-8

中图分类号：

Q5 [生物化学];

学科分类号：

071010 [生物化学与分子生物学]; 081704 [应用化学];

摘要：

Analysis of posttranslational modifications with ubiquitin and ubiquitin-related proteins (Ubl) generally involves detection of the modified species by immunoblotting or autoradiography, techniques that are not easily applicable for kinetic, quantitative, or high-throughput assays. To circumvent these limitations for studies on ubiquitin-related proteins of the SUMO family, we have developed a fluorescence resonance energy transfer (FRET)-based assay system using yellow fluorescent protein (YFP)-tagged mature SUMO1 (amino acids 1-97) and cyan fluorescent protein (CFP)-tagged RanGAP1 (amino acids 400-589) as model substrates. Reactions are set up in 384-well microtiter plates and are followed online using a fluorescence microtiter plate reader. Applications may involve identification and analysis of SUMO-modifying enzymes and isopeptidases, comparison of enzyme and substrate mutants, and screens for small molecular weight inhibitors. The principal outline of the assay should be applicable to other Ubl conjugation systems as well.

引用

页码：20 / 32

页数：13

共 18 条

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Characterization of the localization and proteolytic activity of the SUMO-specific protease, SENP1 [J].