Methods and compositions for tagging via azido substrates
    4.
    发明申请
    Methods and compositions for tagging via azido substrates 审中-公开
    通过叠氮底物标记的方法和组合物

    公开(公告)号:US20060166286A1

    公开(公告)日:2006-07-27

    申请号:US11375635

    申请日:2006-03-14

    IPC分类号: G01N33/53

    摘要: The invention provides methods and compositions for azide tagging of biomolecules. In one embodiment of the invention, proteins are tagged by metabolic incorporation of prenylated azido-analog substrates. Examples of such analogs are azido farnesyl diphosphate and azido farnesyl alcohol. The azido moiety in the resulting modified proteins provides an affinity tag, which can be chemoselectively captured by an azide-specific conjugation reaction, such as the Staudinger reaction, using a phosphine capture reagent. When the capture agent is biotinylated, the resulting conjugates can be detected and affinity-purified by streptavidin-linked-HRP and streptavidin-conjugated agarose beads, respectively. The invention allows detection and isolation of proteins with high yield, high specificity, and low contamination without harsh treatment of proteins.

    摘要翻译: 本发明提供了用于生物分子的叠氮标签的方法和组合物。 在本发明的一个实施方案中,蛋白质通过代谢并入预先化合的叠氮基 - 类似物底物进行标记。 这种类似物的实例是叠氮法呢基二磷酸酯和叠氮法呢醇。 得到的修饰蛋白质中的叠氮基部分提供亲和标签,其可以使用磷化氢捕获试剂通过叠氮化物特异性缀合反应(例如Staudinger反应)进行化学选择性捕获。 当捕获剂被生物素化时,可以分别通过链霉亲和素连接的HRP和链霉抗生物素蛋白缀合的琼脂糖珠分别检测所得的缀合物并进行亲和纯化。 本发明允许以高产量,高特异性和低污染物检测和分离蛋白质,而不严格地处理蛋白质。