Kirley T L
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
Anal Biochem. 1989 Aug 1;180(2):231-6. doi: 10.1016/0003-2697(89)90422-3.
Procedures which allow rapid, quantitative, and selective fluorescent labeling of protein cyst(e)ine residues prior to electrophoresis by reaction with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) under mild conditions are described. After labeling, the protein(s) of interest is easily monitored throughout electrophoresis and subsequent electroblotting or electroelution procedures. The stoichiometry of labeling and therefore the number of cysteine and/or half-cystine residues can be measured spectrophotometrically or fluorometrically and the derived cyst(e)ine adduct can also be quantitated by amino acid analysis and identified in protein sequencing. N-terminal blockage is not observed under the conditions utilized, nor are any other amino acid side chains modified. The procedures described allow complete, rapid, and facile reduction and alkylation of proteins with simultaneous incorporation of a fluorophore, permitting sensitive detection in subsequent manipulation of the proteins. Quantitative fluorescence prelabeling also allows the generation, purification, and sequencing of peptide fragments containing cyst(e)ine residues for determination of internal sequences and residues involved in disulfide bonds.
本文描述了在温和条件下，通过与4-(氨磺酰基)-7-氟-2,1,3-苯并恶二唑(ABD-F)反应，在电泳前对蛋白质半胱氨酸残基进行快速、定量和选择性荧光标记的方法。标记后，在整个电泳以及随后的电印迹或电洗脱过程中，可以轻松监测目标蛋白质。标记的化学计量比以及因此半胱氨酸和/或半胱氨酸残基的数量可以通过分光光度法或荧光法测量，并且衍生的半胱氨酸加合物也可以通过氨基酸分析进行定量，并在蛋白质测序中进行鉴定。在所使用的条件下未观察到N端封闭，也没有其他氨基酸侧链被修饰。所描述的方法允许对蛋白质进行完全、快速和简便的还原和烷基化，同时引入荧光团，从而在后续蛋白质操作中实现灵敏检测。定量荧光预标记还允许生成、纯化和测序含有半胱氨酸残基的肽片段，以确定内部序列和参与二硫键的残基。
