Rösli Christoph, Rybak Jascha-N, Neri Dario, Elia Giuliano
Institute of Pharmaceutical Sciences, Swiss. Federal Institute of Technology, Zurich, Switzerland.
Methods Mol Biol. 2008;418:89-100. doi: 10.1007/978-1-59745-579-4_8.
The strong interaction between streptavidin and biotin is one of the most commonly exploited tools in chemistry and biology. Methods for the facile derivatization of a variety of molecules (in particular, proteins) with biotin have been introduced, in order to allow their efficient recovery, immobilization and detection with streptavidin-based reagents. However, when desired, the release of biotinylated proteins from the streptavidin-based reagents remains a major problem, due to the extraordinary stability of this complex. This chapter presents a protocol developed in our laboratory for the quantitative elution of biotinylated proteins from streptavidin sepharose, featuring harsh elution conditions and competition with free biotin. The usefulness of the method is shown by the recovery of biotinylated proteins from organ homogenates, obtained from mice perfused with a reactive ester derivative of biotin.
链霉亲和素与生物素之间的强相互作用是化学和生物学中最常用的工具之一。为了能够使用基于链霉亲和素的试剂对其进行高效回收、固定和检测，人们已经引入了多种用生物素对各种分子（尤其是蛋白质）进行简便衍生化的方法。然而，在需要时，由于这种复合物具有非凡的稳定性，从基于链霉亲和素的试剂中释放生物素化蛋白质仍然是一个主要问题。本章介绍了我们实验室开发的一种从链霉亲和素琼脂糖中定量洗脱生物素化蛋白质 的方法，该方法具有苛刻的洗脱条件以及与游离生物素的竞争。从用生物素的活性酯衍生物灌注的小鼠获得的器官匀浆中回收生物素化蛋白质，证明了该方法的实用性。
