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Author:
Date:
2017-06-20
[Abstract] This method was generated to isolate high affinity protein complexes from yeast lysate by performing serial affinity purification of doubly tagged 3xFLAG/V5 proteins. First, the bait protein of interest is immunoprecipitated by anti-FLAG-conjugated magnetic beads and gently eluted by 3xFLAG antigen peptide. Next, the bait protein is recaptured from the first eluate by anti-V5-conjugated magnetic beads and eluted with ionic detergent. In this manner, the majority of abundant, nonspecific proteins remain either bound to the first beads or in the first eluate, allowing pure, high affinity complexes to be obtained. This approach can be used to show specific interactions with notoriously ‘sticky’ chaperone proteins.
[摘要] 产生这种方法通过双重标记的3xFLAG / V5蛋白的连续亲和纯化来分离来自酵母裂解物的高亲和力蛋白质复合物。 首先，感兴趣的诱饵蛋白通过抗FLAG缀合的磁珠免疫沉淀，并用3xFLAG抗原肽轻轻洗脱。 接下来，通过抗V5共轭磁珠从第一洗脱液中回收诱饵蛋白质并用离子型洗涤剂洗脱。 以这种方式，大多数丰富的非特异性蛋白质仍然与第一珠粒或第一洗脱液结合，从而获得纯的高亲和性复合物。 这种方法可用于显示与臭名昭着的“粘滞”伴侣蛋白的特定相互作用。
【背景】通过质谱（IP / MS）进行免疫沉淀是鉴定与特定目标诱饵蛋白质的蛋白质 - 蛋白质相互作用的无偏见方法。虽然这种方法被有效地应用于鉴定蛋白质相互作用网络，但是它被假阳性困扰 - ...
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