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Company: Thermo Fisher Scientific
Catalog#: 12-544-7
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Bimolecular Fluorescence Complementation (BiFC) Assay for Direct Visualization of Protein-Protein Interaction in vivo
[Abstract]  Bimolecular Fluorescence Complementation (BiFC) assay is a method used to directly visualize protein-protein interaction in vivo using live-cell imaging or fixed cells. This protocol described here is based on our recent paper describing the functional association of human chromatin adaptor and transcription cofactor Brd4 with p53 tumor suppressor protein (Wu et al., 2013). BiFC was first described by Hu et al. (2002) using two non-fluorescent protein fragments of enhanced yellow fluorescent protein (EYFP), which is an Aequorea victoria GFP variant protein, fused respectively to a Rel family protein and a bZIP family transcription factor to investigate interactions between these two family members in living cells. The YFP was later improved by ... [摘要]  双分子荧光互补(BiFC)测定是用于使用活细胞成像或固定细胞直接观察蛋白质 - 蛋白质相互作用的方法。这里描述的协议是基于我们最近的论文描述人类染色质衔接子和转录辅因子Brd4与p53肿瘤抑制蛋白的功能联系(吴等人。2013年)。 BiFC首先由Hu等人(2002)使用增强的黄色荧光蛋白(EYFP)的两种非荧光蛋白片段描述,所述荧光蛋白是Aequorea victoria GFP变体蛋白,分别融合Rel家族蛋白和bZIP家族转录因子,以调查这两个家庭成员在活细胞之间的相互作用。通过引入突变以降低其对pH和氯离子的敏感性,从而产生称为金星荧光蛋白的超增强YFP,而在37℃下不显示减少的荧光,如通常用EYFP观察到的,YFP被改进(Nagai等al。,2006)。荧光信号通过两个非荧光片段(例如,金黄N末端1-158个氨基酸残基,称为Venus-N)及其C-末端159-239个氨基酸的互补而再生残基,命名为Venus-C;参见图1A和Gully等人,2012; Ding等人,2006; ...