Moos M, Nguyen N Y, Liu T Y
Division of Biochemistry and Biophysics, Food and Drug Administration, Bethesda, Maryland 20892.
J Biol Chem. 1988 May 5;263(13):6005-8.
A method allowing initial sequencing yields of 60-85% to be consistently obtained from samples prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer is described in detail. Conducting electrophoresis at a pH near neutrality is the single most important of the modifications made to earlier procedures, but pre-electrophoresis in the presence of glutathione or sodium thioglycolate and use of Immobilon polyvinylidene difluoride membranes all contribute to the success of the technique. When tryptophan was the NH2 terminus of a protein, the phenylthiohydantoin (PTH)-derivative recovered appeared to be an irreversible oxidation product if pre-electrophoresis was not performed. Following pre-electrophoresis, the PTH-derivative recovered co-migrated with that of unmodified tryptophan, and the recovery was higher. Recovery of methionine as its PTH-derivative was not affected by pre-electrophoresis suggesting that thioglycolate in the electrophoresis buffer during sample separation prevented or reversed oxidation of methionine sulfur but did not protect tryptophan.
详细描述了一种方法，该方法能从通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和电泳转移制备的样品中持续获得60%-85%的初始测序产率。在接近中性的pH值下进行电泳是对早期方法所做的最重要的单一修改，但在谷胱甘肽或巯基乙酸钠存在下进行预电泳以及使用Immobilon聚偏二氟乙烯膜都有助于该技术的成功。当色氨酸是蛋白质的NH2末端时，如果不进行预电泳，回收的苯硫基乙内酰脲（PTH）衍生物似乎是一种不可逆的氧化产物。预电泳后，回收的PTH衍生物与未修饰色氨酸的PTH衍生物共同迁移，且回收率更高。蛋氨酸作为其PTH衍生物的回收率不受预电泳的影响，这表明在样品分离过程中电泳缓冲液中的巯基乙酸盐可防止或逆转蛋氨酸硫的氧化，但不能保护色氨酸。
