用荧光染料或其他标记物标记肽,为研究生物相关相互作用(例如受体-配体结合,1-3、蛋白质结构4-6和酶活性)提供了强有力的工具。

供体和受体标记之间的荧光能量转移(FRET)广泛应用于此类研究。它可通过多种不同的方法进行测定,如淬灭和其他强度测量、供体或受体耗竭动力学、荧光寿命或发射各向异性测量。3-13 已经设计和使用了多种酶底物,14-22部分地基于通过第二标记物淬灭发射,其通过标记物分离以及底物切割猝灭剂而被消除。

标记的肽可以通过修饰分离的肽或通过在固相合成期间掺入标记来制备。有三种策略用来使用染料对肽进行标记:

  1. 在肽合成过程中标记。没有被解封闭步骤损坏的染料被掺入到肽链的氨基末端。
  2. 合成肽可以在特定残基上被共价修饰,并在合成后掺入标记。
  3. 合成肽可以由胺或硫醇反应性蛋白标记物被共价标记。

标记的肽在除去其他保护基团并从树脂中释放之前,荧光基团可以与树脂结合的肽的N-末端缀合。胺反应性荧光基团相对于固定化肽的胺要多使用约5倍摩尔量。反应性荧光素、磺酰罗丹明B、四甲基罗丹明、香豆素、曙红、dabcyl、dabsyl或生物素标记物,以及我们的一些新的atto标记物,应该足够稳定来抵抗严苛的脱保护条件。Dabcyl经常被用作淬灭剂。另一种可能性是使用荧光或发色团标记的氨基酸将标记物掺入肽的特定位点。

标记也可以通过使用生物素化的氨基酸间接实现。例如,如果在肽合成中使用了芴甲氧羰基-赖氨酸(生物素)-OH(货号:73749),则生物素基团允许链霉亲和素或者亲和素缀合物特异性结合到那个位点。有多种荧光团以(链霉)亲和素缀合物的形式供货。

在常规合成程序之后,也可以用常用于蛋白质标记的标记物进行标记。这意味着如果半胱氨酸已用于肽,则主要是胺反应性标记物或硫醇反应性标记物。尽管蛋白质标记的通用标准程序基于靶蛋白质的水性溶液,但在有机溶剂(如DMSO或DMF)中标记肽需要特定的修饰。可以加入三乙胺以确保肽的靶氨基基团被去质子化,这是标记程序所需的。

材料
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