
Recombinant Human Di-Ub (K63-linked) FRET TAMRA Pos3, CF Summary
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins.Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration.The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard.In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
UF-330
Formulation | X mg/ml (X µM) in 50 mM HEPES pH 7.5, 200 mM NaCl, 2 mM DTT |
Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
Stability & Storage: | Protect from light. Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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Background: Di-Ubiquitin
With a predicted molecular weight of 17 kDa, Di-Ubiquitin is composed of two Ubiquitin monomers that are covalently linked through an isopeptide bond, which typically form between a lysine residue of one Ubiquitin molecule and the C-terminal glycine residue of another Ubiquitin molecule (1). Each human Ubiquitin monomer is 76 amino acids (aa) in length and shares 96% and 100% aa identity with yeast andmouse Ubiquitin, respectively (2). Ubiquitin has seven lysine residues that can participate in the formation of poly-Ubiquitin chains. The specific lysine residue used in Ubiquitin conjugation is thought to determine the function of poly-ubiquitination in cellular processes such as protein degradation, signaling, and trafficking (3-8).
Linkage specific di-Ubiquitin is a substrate for enzymes that cleave the isopeptide linkage between two Ubiquitin molecules. These FRET-based DUB substrates with isopeptide bonds between Ubiquitin moieties are superior to gel densitometry assays. Since DUBs recognize and cleave substrates with specific steric conditions, each substrate varies in the position of the fluorophore and quencher and must be empirically tested for individual enzymes.
- Scheffner, M. et al. (1995) Nature 373:81.
- Sharp, P.M. & W.-H. Li (1987) Trends Ecol. Evol. 2:328.
- Behrends, C. & J.W. Harper (2011) Nat. Struct. Mol. Biol. 18:520.
- Greene, W. et al. (2012) PLoS Pathog. 8:e1002703.
- Henry, A.G. et al. (2012) Dev. Cell 23:519.
- Tong, X. et al. (2012) J. Biol. Chem. 287:25280.
- Wei, W. et al. (2004) Nature 428:194.
- Zhang, J. et al. (2012) J. Biol. Chem. 287:28646.
Citations for Recombinant Human Di-Ub (K63-linked) FRET TAMRA Pos3, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products.The data collected includes not only links to publications in PubMed,but also provides information about sample types, species, and experimental conditions.
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- The AMSH3 ESCRT-III-Associated Deubiquitinase Is Essential for Plant ImmunityAuthors: T Schultz-La, A Lenk, K Kalinowska, LK Vestergaar, C Pedersen, E Isono, H Thordal-ChCell Rep, 2018;25(9):2329-2338.e5.Species: Plant - Arabidopsis thalianaSample Types: Recombinant ProteinApplications: Ubiquitination
- Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3Authors: MK Nagel, K Kalinowska, K Vogel, GD Reynolds, Z Wu, F Anzenberge, M Ichikawa, C Tsutsumi, MH Sato, B Kuster, SY Bednarek, E IsonoProc. Natl. Acad. Sci. U.S.A., 2017;0(0):.Species: N/ASample Types: Recombinant ProteinApplications: Bioassay
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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)。
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。
真核生物中有3种依赖DNA的RNA聚合酶,即RNA聚合酶I 、II、 III。RNA聚合酶I主要负责转录rRNA,RNA聚合酶II负责催化合成mRNA。
遗传信息从DNA分子转录到RNA分子中的过程称为转录(transcription)。在真核生物中,最初转录生成的RNA称为不均一核RNA(heterogeneous nuclear RNA,hnRNA)。核内不均一RNA 为存在于真核生物细胞核中的不稳定、大小不均的一组高分子RNA(分子量约为105~2×107,沉降系数约为30-100S)之总称。占细胞全部RNA之百分之几,在核内主要存在于核仁的外侧。认为hnRNA多属信使RNA(messenger ribonucleic acid,mRNA)之前体,包括各种基因的转录产物及其成为mRNA前的各中间阶段的分子,在5'末端多附有间隙结构,而3'的末端附有多聚腺苷酸聚合酶分子。这些hn-RNA在受到加工之后,移至细胞质,作为mRNA而发挥其功能。大部分的hnRNA在核内与各种特异的蛋白质形成复合体而存在着。
前信使RNA(英语:Precursor mRNA,简称为前mRNA)是一种未成熟的单链信使核糖核酸(mRNA)。前mRNA是从细胞核中的DNA模板通过转录而合成的。前mRNA构成了不均一核RNA(或称为核内异质RNA或核内不均一RNA,简称为hnRNA)数量上的主体。hnRNA这一术语通常被用作为前mRNA的同义词,尽管,在严格的意义上说,hnRNA可以包含那些并不最终成为胞浆中mRNA的细胞核RNA转录物。一旦前信使RNA被完全加工完成,他就被叫做“成熟信使RNA”、“成熟mRNA”或被称为“mRNA”。真核前信使RNA在其被加工成为mRNA之前仅短暂地存在。前信使RNA包含有两种片段,外显子与内含子。外显子是在最终的mRNA之中被保留下来的片段,然而内含子则在一步称为剪接的过程中被除去,这一步由剪接体实行(自剪接内含子除外)。
对于真核前信使RNA,附加了对5'与3'修饰的追加加工步骤。其中包括了加上7-甲基鸟苷的5'端帽以及多腺苷酸化。此外,由核小核糖核蛋白颗粒组成的剪接体也会切除真核前信使RNA的内含子。
当一条前信使RNA链被正确地加工为一条信使RNA序列时,它被细胞核输出并最终被翻译成为蛋白质,这是一个由核糖体协力完成的过程。
在原核细胞中,剪接是通过自身催化或内分解切割而完成的。并不涉及蛋白质的自身催化切割仅仅保留用作编码rRNA的那部分,而内分解切割针对于tRNA前体。
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
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
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)。
从地球形成之初的一片混沌到第一个生命的出现,期间究竟发生了哪些事情一直是许多科学家想知道的事情。藉由巴斯德对于自然发生说的驳斥到达尔文的天择说,我们对于生命的起源这个大问题(BigQuestion)的答案也慢慢有越来越多的认识,而透过具有催化功能之RNA的发现,“RNA世界(RNAworld)”的假说也渐渐成为目前解释生命起源里最主流的说法。
尽管RNA同时具有可携带遗传讯息与催化的能力,使其被认为最适合作为早期的生命物质,但在RNA之前的生命物质为何、RNA如何将携带遗传讯息的能力传给目前的DNA及如何将酶催化等功能移转给蛋白质等问题却一直缺乏有力的假说。特别是功能(例如酶活性),由于其系取决于三度空间之排列方式,功能并无法如遗传讯息般,可经由配对(base-pairing)方式有效地在DNA与RNA之间以线性方式传递,造成生物分子间的功能传递机制始终不明。
藉由活体外演化的技术,来自ScrippsResearchInstitute的研究人员成功证明了两种系统间除了遗传讯息可透过一对一对应之方式转移,在一定次数的突变下,功能也有可能在两系统间进行转移。于其实验中,研究人员以R3CRNA连接(由57个核酸所组成的RNA酶)为基础先合成了相对应的DNA序列,如所预期,初合成之DNA序列并不具任何催化活性。然而,在透过一定循环的活体外演化技术后,科学家成功地在试管中发现了一段具有和原始之RNA连接?活性相当的DNA序列,这证明了以核酸为基础之遗传讯息系统之间,除了遗传讯息可透过线性的方式进行传递,在一定次数的突变之下,功能也可以同样的方式在两系统间进行转移。
原学术论文:
NatashaPaul,GregSpringsteen,andGeraldF.Joyce,2006,ConversionofaRibozymetoaDeoxyribozymethroughInVitroEvolution,Chemistry&BIOLOGy,13,p.329–338

