Method for identification of heme-binding proteins and quantification of their interactions
Method for identification of heme-binding proteins and quantification of their interactions
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DOI：
10.1016/j.ab.2020.113865
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发表时间：
2020-10-15
影响因子：
2.9
通讯作者：
Dimitrov, Jordan D.
中科院分区：
文献类型：
--
作者：
Bozinovic, Nina;Noe, Remi;Dimitrov, Jordan D.
表征血红素与蛋白质相互作用的标准方法是吸收光谱。然而，这种方法需要相对大量的蛋白质，很难以高通量的方式进行。在这里，我们描述了一种基于血红素与预涂覆载体的共价原位偶联的免疫吸附试验。这种检测方法的优点是，它只使用最少量的蛋白质(1-10微克)，既可以鉴定血红素结合蛋白，也可以定量测定它们的结合亲和力。重要的是，同样的方法可以用于其他天然或合成化合物的共价连接，并分析它们与蛋白质的相互作用。
The standard assay for characterization of interaction of heme with proteins is absorbance spectroscopy. However, this approach demands relatively large quantities of proteins and it is difficult to perform in high-throughput manner. Here, we describe an immunosorbent assay based on the covalent in situ conjugation of heme to a pre-coated carrier. Advantage of this assay is that it allows both identification of heme-binding proteins and quantification of their binding avidity, using only minimal amounts of protein (1-10 mu g). Importantly, the same approach can be used for covalent linkage of other natural or synthetic compounds and analyzing their interactions with proteins.