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Glass beads (425-600 μm)

玻璃珠,酸洗

Company: Sigma-Aldrich
Catalog#: G8772
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Expression and Purification of Arabidopsis Transmembrane Protein BCM1 in Saccharomyces cerevisiae
Author:
Date:
2020-09-20
[Abstract]  Heterologous expression and purification of transmembrane proteins have remained a challenge for decades hampering detailed biochemical and structural characterization of key enzymes and their interacting regulators in multiple metabolic pathways. An in-depth study on the newly identified Arabidopsis thaliana integral membrane protein BALANCE OF CHLOROPHYLL METABOLISM 1 (BCM1) showed a stimulatory effect of the BCM1 on magnesium chelatase, the first enzyme of chlorophyll biosynthesis, through interaction with the GENOMES UNCOUPLED 4 (Wang et al., 2020). Here, we report a detailed and optimized method for heterologous expression and purification of His-tagged BCM1 in Saccharomyces cerevisiae. Following this method, we obtained native BCM1 used for in vitro ... [摘要]  [摘要 ] 异源表达和公顷跨膜蛋白的纯化VE 仍然几十年来阻碍了关键酶详述生物化学和结构表征一个挑战小号和它们的相互作用调节在多个代谢途径。上新鉴定进行了深入的研究拟南芥拟南芥叶绿素代谢1(BCM1)的整合膜蛋白BALANCE显示一个通过与相互作用对镁螯合,叶绿素生物合成的第一个酶,所述BCM1的刺激效应基因组中脱开4 (王等等人,2020)。这里 ,我们报告了酿酒酵母中His-tagged BCM1异源表达和纯化的详细和优化方法。˚F ollowing这种方法,我们获得用于本机BCM1 体外酶测定的镁螯合(王等人,2020) 。目前,BCM1的结晶研究正在进行中。这个协议可以适于纯化BCM 1一样从用于酶和结构研究真核生物的跨膜蛋白。

[背景 ] 鉴定翻译后单组的lators其指导LY 调制enzym 一个叶绿素合成的酶的抽动活动可以大大提高我们理解的分子机制,通过该植物保持高效叶绿素叶期间LL合成绿化(Brzezowski 等人,2015年)。然而,叶绿素合成酶及其相互作用蛋白的详细生化分析受到体外重组蛋白可用性的限制。我们最近发现一个叶绿素代谢1(BCM1)的翻译后调节平衡,同时刺激小号叶绿素合成和延迟叶绿素分解,日ERE 被授予叶发育过程中的叶绿素稳态(王等人,2020年)。为了检查BCM1对镁螯合酶(MgCh ...

Manganese Superoxide Dismutase Activity Assay in the Yeast Saccharomyces cerevisiae
Author:
Date:
2020-03-05
[Abstract]  Superoxide dismutases (SODs) act as a primary defence against reactive oxygen species (ROS) by converting superoxide anion radicals (O2-) into molecular oxygen (O2) and hydrogen peroxide (H2O2). Members of this enzyme family include CuZnSODs, MnSODs, FeSODs, and NiSODs, depending on the nature of the cofactor that is required for proper activity. Most eukaryotes, including yeast, possess CuZnSOD and MnSOD. This protocol aims at assessing the activity of the yeast Saccharomyces cerevisiae MnSOD Sod2p from cellular extracts using nitroblue tetrazolium staining. This method can be used to estimate the cellular bioavailability of Mn2+ as well as to evaluate the redox state of the cell. [摘要]  [摘要 ] 超氧化物歧化酶(SOD能)充当主防御针对反应性氧物质(ROS)通过转换的超氧阴离子自由基(O 2 - )为分子氧(O 2 )和过氧化氢(H 2 ? 2 )。这种酶的家庭成员包括CuZnSODs ,MnSODs ,FeSODs 和NiSODs ,这取决于是需要适当的活动辅助因子的性质。大多数真核生物,包括酵母,都具有CuZnSOD 和MnSOD 。该协议旨在评估酵母的活性 使用硝基蓝四唑染色法从细胞提取物中提取酿酒酵母MnSOD Sod2p 。该方法可用于估计Mn 2+ 的细胞生物利用度以及评估细胞的氧化还原状态。

[背景 ] 的SODs被定义为减少正常有氧代谢为氧气和过氧化氢期间形成的氧的有害自由基含金属的抗氧化剂酶。:这些酶是基于需要作为辅因子进行适当的酶活性的金属分类CuZnSODs ,MnSODs ,FeSODs ,和NiSODs 。在酿酒酵母中,有两个S OD :CuZn-Sod1p和Mn-Sod2p(Abreu和Cabelli ...

Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae
Author:
Date:
2018-10-05
[Abstract]  Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5’ cap structure, which prevent their identification through the application of widely-used approaches like oligo(dT) purification. Here, we describe a modified cross-linking affinity purification protocol (cCLAP) based on existing cross-linking and immunoprecipitation (CLIP) methods to isolate mRNAs which could be deadenylated, decapped and/or partially degraded in mRNPs, opening the possibility to detect different types of non-coding RNAs (ncRNAs). Once isolated, ... [摘要]  核糖核蛋白颗粒(mRNP)是由mRNA和RNA结合蛋白(RBP)组成的复合物,其控制mRNA转录定位,转换和翻译。已显示mRNP内的一些mRNA经历降解或储存。那些转录物可能缺乏一般的mRNA元件,如poly(A)尾或5'帽结构,这通过应用广泛使用的方法如oligo(dT)纯化来阻止它们的鉴定。在这里,我们描述了基于现有的交联和免疫沉淀(CLIP)方法的修饰的交联亲和纯化方案(cCLAP),以分离mRNP中可被去腺苷酸化,去除和/或部分降解的mRNA,从而开启了检测不同的可能性。非编码RNA(ncRNA)的类型。分离后,将RNA进行衔接子连接,然后进行下一代测序(NGS)。由于快速有效的交联和淬灭步骤,该方案也适用于瞬时诱导的mRNP颗粒。实例包括由外在应激物触发的处理体(PB)或应力颗粒(SG)。其重现性和广泛应用使该方案成为研究特定RNP的RNA组成的有用且有力的工具。
【背景】mRNP内转录物的表征对于理解细胞转录和转录后过程至关重要。通过交联和免疫沉淀,然后通过RNA-Seq从mRNP颗粒中分离RNA已经成为鉴定mRNA靶标的常用方法(Tagwerker et al。,2006; Hafner et al。,2010; Kishore et al。,2011)。 ...

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