| Bimolecular Fluorescence Complementation (BiFC) for Studying Sarcomeric Protein Interactions in Drosophila
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Author:
Date:
2020-04-05
[Abstract] Protein-protein interactions in Drosophila myofibrils are essential for their function and formation. Bimolecular Fluorescence Complementation (BiFC) is an effective method for studying protein interactions and localization. BiFC relies on the reconstitution of a monomeric fluorescent protein from two half-fragments when in proximity. Two proteins tagged with the different half-fragments emit a fluorescent signal when they are in physical contact, thus revealing a protein interaction and its spatial distribution. Because myofibrils are large networks of interconnected proteins, BIFC is an ideal method to study protein-protein interactions in myofibrils. Here we present a protocol for generating transgenic flies compatible with BiFC and a method for analyzing protein-protein ...
[摘要] [摘要] 果蝇肌原纤维中的蛋白质-蛋白质相互作用对其功能和形成至关重要。双分子荧光互补（BiFC ）是研究蛋白质相互作用和定位的一种有效方法。BiFC 依赖于邻近时从两个半片段重构单体荧光蛋白。标记有不同半片段的两种蛋白质在物理接触时会发出荧光信号，从而揭示了蛋白质相互作用及其空间分布。因为肌原纤维相互连接的蛋白质的大型网络中，附设是一种理想的方法来 研究肌原纤维中蛋白质之间的相互作用。在这里，我们提出了一种生成与BiFC 兼容的转基因果蝇的协议，以及一种基于肌原纤维中荧光BiFC 信号的蛋白质-蛋白质相互作用分析方法。我们的方案适用于大多数果蝇蛋白，只需稍加修改即可用于研究任何组织。
[背景] 肉瘤是横纹肌中最小的收缩单位，并沿着肌原纤维的长度以重复的方式延伸（Reedy和Beall，1993）。肉瘤产生肌肉收缩的能力取决于两个肌原纤维成分：细丝和粗丝。肌球蛋白粗丝固定在肌节中心的M线，而肌动蛋白细丝固定在肌节两侧的Z盘上。因此，Z盘对于维持肌原纤维的结构和收缩至关重要，而Z盘无法形成可能导致各种人类肌病的严重缺陷性肌肉表型（Lemke和Schnorrer，2017年）。
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| MNase Digestion for Nucleosome Mapping in Neurospora
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Author:
Date:
2016-06-05
[Abstract] Digestion of chromatin by micrococcal nuclease MNase followed by high throughput sequencing allows us to determine the location and occupancy of nucleosomes on the genome. Here in this protocol we have described optimized conditions of MNase digestion of filamentous fungus Neurospora crassa chromatin without a requirement of a nuclear fractionation step.
[摘要] 通过微球菌核酸酶MNase消化染色质，然后高通量测序允许我们确定核小体在基因组上的位置和占据。 在这个协议中，我们描述了MNase消化丝状真菌粗糙链孢霉染色体的优化条件，而不需要核分馏步骤。
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| Purification and Sequencing of DNA Guides from Prokaryotic Argonaute
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Author:
Date:
2014-11-20
[Abstract] Some proteins utilize nucleic acids to guide them to complementary nucleic acid targets. One example is prokaryotic Argonaute protein, which, binds small single stranded DNA molecules as guides (Swarts et al., 2014). This protocol describes a method to purify DNA guides from these proteins. It also describes a PCR-based method to enrich the guides by PCR amplification. This methods relies on addition of a poly-A tail at the 3’-end of the ssDNA molecules by Terminal Deoxynucleotidyl Transferase (TdT), followed by ligation of a oligonucleotide to the 5’-end of the ssDNA molecule using T4 RNA ligase, and amplification by PCR. The generated dsDNA products are suitable for traditional cloning and sequencing and high-throughput sequencing. Importantly, the information which strand ...
[摘要] 一些蛋白利用核酸来将它们引导至互补核酸靶。 一个实例是原核Argonaute蛋白，其结合小的单链DNA分子作为指导（Swarts等人，2014）。 该协议描述了从这些蛋白质中纯化DNA指南的方法。 它还描述了基于PCR的方法以通过PCR扩增来富集指南。 该方法依赖于通过末端脱氧核苷酸转移酶（TdT）在ssDNA分子的3'末端添加聚腺苷酸尾，然后使用T4 RNA连接酶将寡核苷酸连接到ssDNA分子的5'末端， 和通过PCR扩增。 所产生的dsDNA产物适合于传统的克隆和测序以及高通量测序。 重要的是，与ssDNA分子匹配的链的信息在该过程中不会丢失。
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