{{'Search' | translate}}
 

Acid-washed glass beads

玻璃珠,酸洗

Company: Sigma-Aldrich
Catalog#: G8772
Bio-protocol()
Company-protocol()
Other protocol()

Polyamine Transport Assay Using Reconstituted Yeast Membranes
Author:
Date:
2021-01-20
[Abstract]  

ATP13A2/PARK9 is a late endo-/lysosomal P5B transport ATPase that is associated with several neurodegenerative disorders. We recently characterized ATP13A2 as a lysosomal polyamine exporter, which sheds light on the molecular identity of the unknown mammalian polyamine transport system. Here, we describe step by step a protocol to measure radiolabeled polyamine transport in reconstituted vesicles from yeast cells overexpressing human ATP13A2. This protocol was developed as part of our recent publication (van Veen et al., 2020) and will be useful for characterizing the transport function of other putative polyamine transporters, such as isoforms of the P5B transport ATPases.

[摘要]  [摘要] ATP13A2 / PARK9是一种晚期内/溶酶体P5B转运ATPase,与多种神经退行性疾病有关。我们最近将ATP13A2表征为溶酶体多胺出口者,这为未知的哺乳动物多胺转运系统的分子身份提供了线索。在这里,我们逐步描述了从过量表达人ATP13A2的酵母细胞中测量重组囊泡中放射性标记的多胺转运的方案。该方案是我们最新出版物的一部分(van Veen等,2020),将有助于表征其他假定的多胺转运蛋白的转运功能,例如P5B转运ATPase的同工型。


[背景] ATP13A2 / PARK9编码一种普遍表达的晚期内-/溶酶体膜蛋白,与一系列神经退行性疾病有关,例如早发性帕金森氏病(Di Fonzo等,2007 ;Lin等,2008)和Kufor -Rakeb综合征(伴痴呆的早期帕金森病)(Ramirez等,2006 ;Park等,2011)。ATP13A2属于P型转运ATPase ,是一类活性转运蛋白,由于ATP水解而暂时形成磷酸中间产物(Kuhlbrandt ,2004年)。ATP13A2是P5亚家族的成员,该家族已在20多年前通过基因组测序鉴定出来(Axelsen和Palmgren ...

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 ...

Comments