S. aureus Gyrase
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S. aureus Gyrase
Staphylococcus aureus gyrase is a bacterial type II topoisomerase which can introduce negative supercoils into DNA. It is a target for both quinolone and coumarin drugs and can be used for screening potential antibacterial compounds. It is prepared by overexpressing the subunits in E. coli and then purified.
It is supplied as an A2B2 complex in Dilution Buffer. Store at -80 °C.
All enzyme is supplied with 5X concentrated Assay Buffer and Dilution Buffer which are also available separately. 1 U of gyrase will supercoil 0.5 µg relaxed pBR322 DNA in 30 minutes at 37 °C.
See technical documents below for more detailed information and lot specific activities.
Technical Documents
S. aureus Gyrase Supercoiling Assay Kits
Kits are available containing everything required to perform supercoiling reactions and to test inhibitors. They contain the enzyme, relaxed DNA substrate and the Assay and Dilution buffers.
Technical Documents
S. aureus Gyrase Cleavage Assay Kits
Some drugs interrupt the DNA breakage-reunion step of the gyrase reaction. This leads to cell death and it is the mechanism behind the action of the quinolones such as nalidixic acid and ciprofloxacin. Cleavage assays are particularly useful in determining if a potential drug acts by this mechanism.
These kits are designed specifically for cleavage reactions. They contain the supercoiled pBR322 DNA substrate and the Assay and Dilution buffers required for DNA cleavage reactions in addition to the S. aureus gyrase and linearised pBR322 marker.
Cleavage specific enzyme available separately on request.
Technical Documents
S. aureus Gyrase Assay Kits for Cell Extracts
These kits are designed for assaying cell extracts containing gyrase and partially purified fractions and contain relaxed DNA substrate, Assay buffer, Dilution buffer and control supercoiled DNA.
Technical Documents
S. aureus Gyrase ATPase Kits
The coumarin drugs inhibit the action of gyrase by competitively inhibiting the hydrolysis of ATP thus preventing supercoiling. These kits can be used to test the effects of potential ATPase inhibitors.
These kits are advantageous because the assays are microtitre plate-based and thus large numbers of compounds can be screened in a relatively short period of time.
Technical Documents
High / Medium-Throughput Assay Kit - S. aureus Gyrase
The kit is supplied with sufficient S. aureus gyrase enzyme, plasmid DNA substrate, buffers and other assay components* for 100 assays. The enzyme is supplied at a concentration of 10 U/μl in Dilution Buffer. The kit is also supplied with sufficient wash buffers for one 96-well plate. These buffers are supplied as 20X concentrates and must be diluted with ultra pure water prior to use.
More information about this assay can be found on the "Services" page under "High/Medium Throughput Assay".
Kit issued with limited licence for individual use only.
Patent held by Inspiralis Ltd., Norwich, Norfolk, UK. (Patent No. GB0424953.8, US7838230)
Technical Documents
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RNA聚合酶的作用位点:3'-5'-磷酸二酯键
DNA解旋酶的作用位点:DNA中互补碱基之间的氢键
2、异硫氰酸胍:目前认为是最有效的RNA酶抑制剂,它在裂解组织的同时也使RNA酶失活,它既可破坏细胞结构使核酸从核蛋白中解离出来,又对RNA酶有强烈的变性作用。
3、氧钒核糖核苷复合物:由氧化钒离子和核苷形成的复合物,它和RNA酶结合形式过渡类物质,几乎能完全抑制RNA酶的活性。
4、RNA酶的蛋白质抑制剂(RNasin):从大鼠肝或人胚盘中提取得来的酸性糖蛋白.Rnasin是RNA酶的一种非竞争性抑制剂,可以和多种RNA酶结合,使其失活。
5、其他:SDS、尿素、硅藻土等对RNA酶也有一定的抑制作用。
不过这种类似的RNA酶清除剂是一种广泛的蛋白灭活剂,如果你配置处理的溶液要用于类似一些酶蛋白的溶液体系,那就不可以用RNA酶清除剂来处理了,否则会影响需要用到的酶的活性
有DNase、RNase、核酸酶S1等,可水解相应的DNA和RNA,核酸酶S1可降解单链DNA和RNA,用量增大也可降解双链核酸。它可用于切去ds-cDNA合成中产生的发夹环。
(2)修饰酶
有些酶可在其他酶的作用下,将酶的结构进行共价修饰,使该酶活性发生改变,
核酶的催化功能与其空间结构有密切关系。
不同的核酶可分为两类:
1 剪切型核酶:只剪不接,如M1 RNA。
2 剪接型核酶:该酶具有序列特异的内切核酸酶、RNA连接酶等多种酶活性。
加工场所在生物体细胞质(或相关实验设备中)。
转录后修饰在生命体中广泛存在(已发现100多种),而假尿嘧啶RNA修饰就是其中最主要的一种。假尿嘧啶在多种非编码RNA(tRNA、rRNA、snRNA等)上的功能与机制已有较多研究;然而,对于信使RNA(messengerRNA,mRNA)上假尿嘧啶的分布和潜在生物学功能,目前知之甚少。最主要的难题,就是如何实现假尿嘧啶在mRNA上的精准定位。
为了研究哺乳动物转录组中的假尿嘧啶修饰,伊成器课题组首先利用高分辨质谱对mRNA中的假尿嘧啶修饰进行定量,发现其广泛存在于各种细胞系及小鼠的组织当中,并且在哺乳动物mRNA中丰度相当高。该研究继而通过化学生物学、高通量测序等手段,发展了“CeU-Seq”—一种利用小分子化合物实现特异性标记与富集的假尿嘧啶高通量测序技术。利用这一技术,该研究成功实现了人细胞系以及小鼠(大脑与肝脏组织)全转录组水平的单碱基分辨率假尿嘧啶检测,发现在数千个mRNA与长非编码RNA(lncRNA)上都含有假尿嘧啶修饰。该研究进一步确定了多个可以作用于mRNA上的假尿嘧啶合成酶(其中PUS1、DKC1两种酶之前被发现与线粒体肌病、先天性角化不良等人类疾病相关),并且发现转录组中假尿嘧啶的含量与分布均会受到各种环境刺激的调控,呈现出“刺激条件特异性”的诱导修饰。因此,该研究不但揭示了假尿嘧啶的广泛存在、绘制了转录组中假尿嘧啶RNA修饰的高清谱图,也为这一转录后修饰参与基因表达调控的研究提供了重要工具、为近年来兴起的“RNA表观遗传学”领域提供了崭新的研究方向。
北京大学生科院博士生李笑雨(PTN-BBS联合培养项目)、生命中心博士生朱平、博士生马士清是这篇论文的并列第一作者,生命科学学院伊成器研究员是该论文的通讯作者。该研究得到了国家自然科学基金、科技部973计划和北大清华生命科学联合中心的资助。
原文链接:http://www.nature.com/nchembio/journal/vaop/ncurrent/full/nchembio.1836.html
详情:http://www.bioku.cn/201212/science-cirp-circADIan-gene-clip-posttranscriptional-modification-fibroblast/
在哺乳动物组织中,节律基因的表达受到局部的振荡器或视交叉上核中生物钟主钟控制的系统信号的调控。本研究发现是受刺激的体温循环而不是外周振荡器,控制纤维母细胞中的冷诱导RNA结合蛋白(CIRP)的节律性表达。反过来,功能缺失性实验表明CIRP是高振幅的节奏基因表达所需的。利用基于生物素-链亲和素的交联和免疫沉淀反应(CLIP),本研究对CIRP结合的RNA进行了全转录组分析,发现了一些编码昼夜节律振荡器蛋白的转录物,包括CLOCK蛋白。此外,在CIRP缺失的纤维母细胞中,CLOCK的含量大大减少。因为在这些细胞中,CLOCK的异位表达提高了节律基因的表达水平,因此我们猜测CIRP通过调节CLOCK的表达水平赋予了昼夜节奏振荡器的健壮性。
比如用QIAGEN的RNase-free的DNase处理的,不是加了DNase就结束了,为了去除DNase(蛋白)和buffer等可能影响到后期操作的因素,所以酶解了DNA后必须纯化RNA(QIAGEN的纯化柱),这样得到的RNA才用于定量实验。即使这样,在做real-time PCR时都要加一管仅加RNA的作为阴性对照(排除DNA的污染)。