| Biochemical Isolation of Myonuclei from Mouse Skeletal Muscle Tissue
|
|
Author:
Date:
2017-12-20
[Abstract] Skeletal muscle provides the contractile force necessary for movement, swallowing, and breathing and, consequently, is necessary for survival. Skeletal muscle cells are unique in that they are extremely large cells containing thousands of nuclei. These nuclei must all work in concert to maintain skeletal muscle function and thereby maintain life. The nucleus is a major site of signaling integration and gene expression regulation. However, examining nuclear processes in skeletal muscle can be difficult because myonuclei are challenging to isolate. We optimized a protocol to purify myonuclei from whole muscle tissue using ultracentrifugation over a discontinuous sucrose gradient to separate the nuclear fraction. We used these purified nuclei for downstream applications including flow ...
[摘要] 骨骼肌提供运动，吞咽和呼吸所需的收缩力，因此是生存所必需的。骨骼肌细胞是独特的，因为它们是含有数千个核的极大细胞。这些细胞核必须协调一致才能维持骨骼肌功能，从而维持生命。细胞核是信号整合和基因表达调控的主要部位。然而，检查骨骼肌中的核过程可能是困难的，因为肌核难以分离。我们优化了一个协议，从整个肌肉组织使用超速离心蔗糖梯度来分离核分数的纯化肌细胞。我们将这些纯化的细胞核用于下游应用，包括流式细胞术和质谱。我们使用这种方法来比较年轻和老鼠后肢肌肉的肌细胞蛋白质组（Cutler等人，2017）。这个协议可能适用于分离myonuclei的各种下游分析，如流式细胞仪，显微镜，蛋白质印迹和蛋白质组学。
【背景】为了生存，必须保持适当的骨骼肌功能。这种维持的一个组成部分是根据细胞需要和环境线索调整基因表达。调节基因表达的核过程是调节细胞组成和行为的关键组成部分。然而，由于四个技术限制，涉及这些过程的肌细胞蛋白难以研究。首先，骨骼肌是致密的，紧密地填满了组织中超过60％的蛋白质的收缩性蛋白质（Deshmukh等人，2015; Cutler等人）。 ...
|
|
|
| Sample Preparation of Telomerase Subunits for Crystallization
|
|
Author:
Date:
2015-08-20
[Abstract] Telomerase is a large ribonucleoprotein complex that replicates the linear chromosome ends in most eukaryotes. Large-scale preparation of the telomerase core components in vitro has long been a big challenge in this field, hindering the understanding of the catalytic mechanism of telomerase, as well as slowing down the development of telomerase inhibitors for cancer therapy. We have successfully developed a protocol for large-scale preparation of the TRBD-CR4/5 complex of the medaka telomerase in vitro, and used this method to study the high-resolution structure of the TRBD-CR4/5 complex by X-ray crystallography. This procedure may be also adapted to purify other protein-RNA complexes for structural studies.
[摘要] 端粒酶是在大多数真核生物中复制线性染色体末端的大核糖核蛋白复合物。 体外大规模制备端粒酶核心组分长期以来一直是该领域的一个大挑战，阻碍了对端粒酶的催化机理的理解，以及减慢端粒酶抑制剂对癌症的发展 治疗。 我们成功地开发了用于大规模制备体外的medaka端粒酶的TRBD-CR4/5复合物的方案，并且使用该方法研究TRBD-CR4/5复合物的高分辨率结构， 5复合物通过X射线晶体学。 该程序也可以适于纯化其它蛋白质-RNA复合物用于结构研究。
|
|
|