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Bromophenol Blue sodium salt

溴酚蓝钠盐

Company: Sigma-Aldrich
Catalog#: B5525
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Chromatin Immunoprecipitation Experiments from Whole Drosophila Embryos or Larval Imaginal Discs
Author:
Date:
2017-06-05
[Abstract]  Chromatin Immunoprecipitation coupled either to qPCR (qChIP) or high-throughput sequencing (ChIP-Seq) has been extensively used in the last decades to identify the DNA binding sites of transcription factors or the localization of various histone marks along the genome. The ChIP experiment generally includes 7 steps: collection of biological samples (A), cross-linking proteins to DNA (B), chromatin isolation and fragmentation by sonication (C), sonication test (D), immunoprecipitation with antibodies against the protein or the histone mark of interest (E), DNA recovery (E), identification of factor-associated DNA sequences by PCR or sequencing (F). The protocol described here can readily be used for ChIP-seq and ChIP-qPCR experiments. The entire procedure, describing experimental setup ... [摘要]  与qPCR(qChIP)或高通量测序(ChIP-Seq)相结合的染色质免疫沉淀已被广泛用于识别转录因子的DNA结合位点或基因组中各种组蛋白标记的定位。 ChIP实验通常包括7个步骤:收集生物样品(A),交联蛋白质到DNA(B),染色质分离和通过超声处理分离(C),超声处理测试(D),用针对蛋白质的抗体进行免疫沉淀感兴趣的组蛋白标记(E),DNA回收(E),通过PCR或测序鉴定因子相关DNA序列(F)。这里描述的协议可以容易地用于ChIP-seq和ChIP-qPCR实验。描述在完整的果蝇组织中优化分析的实验设置条件的整个过程可以在四天内完成。

背景 尽管永生化的培养细胞广泛用于研究各种细胞类型的染色质景观,但是在生理条件下在体内探测相互作用的有价值的方法对于进行转录的时间或空间比较分析是必要的因子和组蛋白修饰图在不同阶段的果蝇发展或不同组织之间。在这里,我们提供了一个详细的ChIP协议,已被优化,以便在整个果蝇胚胎和幼虫成像光盘上工作,突出关键的实验参数。

Chase Assay of Protein Stability in Haloferax volcanii
Author:
Date:
2017-03-20
[Abstract]  Highly regulated and targeted protein degradation plays a fundamental role in almost all cellular processes. Determination of the protein half-life by the chase assay serves as a powerful and popular strategy to compare the protein stability and study proteolysis pathways in cells. Here, we describe a chase assay in Haloferax volcanii, a halophilic archaeon as the model organism. [摘要]  高度调节和靶向的蛋白质降解在几乎所有的细胞过程中发挥重要作用。通过追踪测定法测定蛋白质半衰期是比较蛋白质稳定性和研究细胞中蛋白水解途径的有力和普遍的策略。在这里,我们描述了作为模型生物体的嗜盐古细菌Haloferax volcanii 中的追踪测定。

背景 在真核生物中,泛素蛋白酶体系统在高选择性和靶向性蛋白水解中起主要作用(Glickman and Ciechanover,2002)。最近的证据表明,小型古细菌泛素样修饰蛋白或SAMPs也起到了蛋白酶体破坏的靶向蛋白的作用(Maupin-Furlow,2014; Anjum等人,2015; Fu et al。 。,2016)。体内蛋白质半衰期的测量提供了研究蛋白水解途径的直接途径。环己酰胺追踪和脉冲追踪测定通常用于监测真核生物中靶向蛋白质的降解(Zhou,2004)。前一种方法用于确定放线菌酮抑制平移伸长后所有细胞蛋白的半衰期;而脉冲追踪测定法测量新合成(脉冲标记)蛋白质的周转率,而不干扰正常的细胞生长。与真核系统相比,确定古细菌中一种给定蛋白质半衰期的快速简便方法尚未明确。因此,我们开发了一种方案,用于测量盐爱好的考古Haloferax volcanii的体内蛋白质稳定性。使用翻译抑制剂(茴香霉素)和转录(放线菌素D)来最小化该古细菌中新蛋白质的合成。 ...

Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate
Author:
Date:
2016-10-05
[Abstract]  This protocol describes the in vitro phosphorylation of ubiquitin and Parkin by the kinase PINK1 using recombinant proteins. Both substrates, ubiquitin and Parkin, are phosphorylated at the conserved serine 65 residue (pS65-ubiquitin and pS65-Parkin). The protocol also includes the use of monomeric and K48- and K63-linked poly-ubiquitin chains as alternative substrates. Although there are commercially available antibodies, we have not tested their performance in this assay since, but used validated antibodies from our laboratory. An alternative antibody-independent method, the use of phos-tag gels to detect pS65-ubiquitin and pS65-Parkin, is described in addition. [摘要]  该协议描述了通过激酶PINK1使用重组蛋白的泛素和帕金蛋白的体外磷酸化。两种底物,泛素和帕金蛋白,在保守的丝氨酸65残基(pS65-泛素和pS65-帕金蛋白)上磷酸化。该方案还包括使用单体和K48和K63连接的聚泛素链作为替代底物。虽然有市售的抗体,我们没有测试他们在这个测定中的性能,因为,但使用我们实验室的验证抗体。另外描述了另一种抗体非依赖性方法,使用phos-标记凝胶检测pS65-泛素和pS65-帕金。

[背景] 在细胞中, PINK1是稳定和激活的线粒体膜去极化和其他形式的应力,导致线粒体损伤。活化的PINK1磷酸化泛素,其作为线粒体表面上胞质E3泛素连接酶Parkin的受体。 Parkin对PINK1的磷酸化是Parkin对线粒体底物的完全活性所必需的。活性pS65-Parkin的存在在前馈机制中扩增了作为线粒体标记的线粒体上的pS65-泛素的量。最终,受损的线粒体被自噬噬菌体衔接子识别,并将被蛋白酶体和自噬(mitophagy)降解。这种关键的线粒体质量控制通路促进线粒体的周转,并防止可导致细胞变性的功能障碍线粒体的积累。 PINK1或Parkin中的功能缺失突变与早发性帕金森病相关。
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