| Detection of Protein Interactions in the Cytoplasm and Periplasm of Escherichia coli by Förster Resonance Energy Transfer
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Author:
Date:
2018-01-20
[Abstract] This protocol was developed to qualitatively and quantitatively detect protein-protein interactions in Escherichia coli by Förster Resonance Energy Transfer (FRET). The described assay allows for the previously impossible in vivo screening of periplasmic protein-protein interactions. In FRET, excitation of a donor fluorescent molecule results in the transfer of energy to an acceptor fluorescent molecule, which will then emit light if the distance between them is within the 1-10 nm range. Fluorescent proteins can be genetically encoded as fusions to proteins of interest and expressed in the cell and therefore FRET protein-protein interaction experiments can be performed in vivo. Donor and acceptor fluorescent protein fusions are constructed for bacterial proteins ...
[摘要] 该协议的开发是通过Förster共振能量转移（FRET）定性和定量检测大肠杆菌中的蛋白质 - 蛋白质相互作用。所描述的测定允许以前不可能的周质蛋白质 - 蛋白质相互作用的体内筛选。在FRET中，供体荧光分子的激发导致能量转移到受体荧光分子，如果它们之间的距离在1-10nm范围内，则受体荧光分子将发光。荧光蛋白质可以被遗传编码为与感兴趣的蛋白质的融合物并且在细胞中表达，因此FRET蛋白质 - 蛋白质相互作用实验可以在体内进行。供体和受体荧光蛋白融合体被构建用于被怀疑相互作用的细菌蛋白质。这些融合蛋白在细菌细胞中共表达，随后激发供体和受体通道测量荧光发射光谱。供体的发射光谱与受体的激发光谱之间的部分重叠是FRET的先决条件。即使在没有FRET的情况下，供体激发也可以使受体以已知百分比交叉激发。通过测量背景，仅供体和仅受体样品的参考光谱，可以计算预期的发射光谱。在预期光谱之上的受体的致敏发射可以归因于FRET，并且可以通过光谱解混来量化。
【背景】确定如何和哪些蛋白质相互作用维持生命是分子生物学研究的核心。存在许多体外方法，但可能导致误报，因为相互作用是从其生物学背景中取出的。 ...
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| Exit from Pluripotency Assay of Mouse Embryonic Stem Cells
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Author:
Date:
2017-08-20
[Abstract] A novel method to assess the dissolution of the core pluripotency transcription-factor circuit of mouse Embryonic Stem Cells (mESCs) has been developed (Ying et al., 2003; Betschinger et al., 2013). In order to efficiently identify genes essential for the break-down of the pluripotency network in mutant mESCs with proliferation defects, we adapted this ‘exit from pluripotency assay’ (Bodak et al., 2017; Cirera-Salinas et al., 2017). The protocol described here has been successfully applied to several mESC lines and is easily transposable from one laboratory to another.
[摘要] 已经开发了评估小鼠胚胎干细胞（mESCs）的核心多能转录因子电路溶解的新方法（Ying等，2003; Betschinger等，2013）。 为了有效识别具有增殖缺陷的突变体mESCs中多能网络分解所必需的基因，我们调整了这种“多能性测定法”（Bodak等，2017; Cirera-Salinas等，2017）。 这里描述的方案已经成功应用于几个mESC系列，并且可以容易地从一个实验室转座到另一个实验室。
【背景】几十年来，科学家已经尝试确定基因与一般（例如胚胎体）或定向（例如，神经元前体细胞）分化方案的mESCs的分化潜能的机制。最近，发现2i培养基允许在体外俘获天真的干细胞（Ying et al。，2008）。 ...
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| Immunolabeling of Proteins in situ in Escherichia coli K12 Strains
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Author:
Date:
2013-08-05
[Abstract] This protocol was developed to label proteins in bacterial cells with antibodies conjugated to a fluorophore for fluorescence microscopy imaging. The procedure is optimized to minimize morphological changes and also to minimize the amount of antibodies needed for the staining. The protocol can also be used with primary antibodies conjugated to a fluorophore. The method has been verified extensively (van der Ploeg et al., 2013), but it should be noted that one case in Caulobacter crescentus (Hocking et al., 2012) has been reported in which the localization of a protein changed upon fixation by formaldehyde/glutaraldehyde. However, the localization of the same protein in E. coli did not change.
[摘要] 该协议被开发来标记细菌细胞中的蛋白质与缀合到荧光显微镜成像荧光的抗体。 优化程序以使形态变化最小化，并且使染色所需的抗体的量最小化。 该方案还可以与结合至荧光团的一抗一起使用。 该方法已经广泛地验证（van der Ploeg等人，2013），但是应当注意的是，在新月柄杆菌中的一种情况（Hocking等人，/2012>），其中蛋白质的定位在通过甲醛/戊二醛固定时改变。 然而，相同蛋白质在E中的定位。 大肠杆菌没有改变。
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