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cOmpleteTM, Mini Protease Inhibitor Cocktail

蛋白酶抑制剂混合物,不含EGTA

Company: Roche Diagnostics
Catalog#: 11836153001
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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)。 ...

Streptavidin Bead Pulldown Assay to Determine Protein Homooligomerization
Author:
Date:
2017-11-20
[Abstract]  Pulldown assay is a conventional method to determine protein-protein interactions in vitro. Expressing a protein of interest with two different tags allows testing whether both versions can be captured via one of the two tags as homooligomeric complex. This protocol is based on streptavidin bead capture of a biotinylated protein and co-associated Flag-tagged protein using Streptavidin MagBeads. [摘要]  Pulldown分析是一种常规的方法来确定蛋白质在体外的相互作用。 用两种不同的标签表达感兴趣的蛋白质可以检测两种标签是否可以通过两种标签之一作为同低聚体复合物来捕获。 该方案基于使用链霉抗生物素蛋白MagBeads的链霉抗生物素蛋白珠捕获生物素化蛋白质和共结合Flag标记蛋白质。
【背景】淀粉样前体蛋白(APP)可以通过其大的胞外结构域及其跨膜结构域形成同型二聚体,在生物学功能中起重要作用。 目前的方案已被用于表征APP跨膜C-末端99个氨基酸片段(C99)的同二聚化(Yan等人,2017)。 该检测的基本原理如图1所示:链霉亲和素包被的MagBeads可以捕获生物素化的蛋白质,这可以拉下相互作用蛋白质,并通过抗FLAG抗体检测。

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图1.基于MagBeads的pull-down测定的原理在该特定的方案中,使用生物素化的Avi-标记的C99蛋白和相关的C99-TEV位点-rTA-Flag蛋白质。

Investigating the Assembly Status of the Plastid Encoded Polymerase Using BN-PAGE and Sucrose Gradient Centrifugation
Author:
Date:
2016-07-20
[Abstract]  The plastid encoded polymerase (PEP) represents a major transcription machinery in mature chloroplasts (Liere et al., 2011; Zhelyazkova et al., 2012). The proper assembly of this multi-subunit complex is important for plant growth and development (Pfalz and Pfannschmidt, 2013). The PEP polymerase can be purified from soluble and from membrane-bound (also named transcriptionally active chromosome, TAC) fractions. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) and sucrose gradient sedimentation followed by immunoblot analyses is used to detect the status of the PEP complex assembly. [摘要]  质体编码聚合酶(PEP)代表成熟叶绿体中的主要转录机制(Liere等人,2011; Zhelyazkova等人,2012)。 这种多亚基复合物的正确装配对于植物生长和发育是重要的(Pfalz和Pfannschmidt,2013)。 PEP聚合酶可以从可溶性和膜结合(也称为转录活性染色体,TAC)级分中纯化。 蓝色使用天然聚丙烯酰胺凝胶电泳(BN-PAGE)和蔗糖梯度沉淀,随后进行免疫印迹分析来检测PEP复合物组装体的状态。

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