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Anti-FLAG® M2 Magnetic Beads

M2磁珠

Company: Sigma-Aldrich
Catalog#: M8823
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Protein Expression and Purification of the Hsp90-Cdc37-Cdk4 Kinase Complex from Saccharomyces cerevisiae
Author:
Date:
2017-10-05
[Abstract]  Interactions between Hsp90, its co-chaperone Cdc37 and kinases have been biochemically studied for over three decades and have been shown to be functionally important in organisms from yeast to humans. However, formation of a stable complex for structural studies has been elusive. In this protocol we describe expression and purification of Hsp90-Cdc37-Cdk4 kinase protein complex from Saccharomyces cerevisiae utilizing the viral 2A sequences to titrate the three proteins at similar levels. [摘要]  Hsp90,其伴侣伴侣Cdc37和激酶之间的相互作用已经在三十多年的生物化学研究中被证明在酵母与人类的生物体内在功能上是重要的。 然而,形成一个稳定的结构研究复合物是难以捉摸的。 在该方案中,我们描述了利用病毒2A序列以相似水平滴定三种蛋白质的来自酿酒酵母的Hsp90-Cdc37-Cdk4激酶蛋白复合物的表达和纯化。
【背景】Hsp90分子伴侣与其客体激酶之间的稳定形成复合物已经被证明在体外是难治性的。以前的工作表明,Hsp90的共伴伴Cdc37与昆虫Sf9细胞中的客体激酶的过表达导致Sf9 Hsp90,外源Cdc37和外源激酶(Vaughan等人,2006)之间的稳定复合物。然而,昆虫细胞培养需要特殊的设备,比其他研究较好的表达系统(如细菌和酵母)要难以进行遗传操作,并且显着较慢地生长和克隆。上述蛋白质在E中的共表达。大肠杆菌不产生可溶性激酶/稳定复合物。我们认为,酿酒酵母将具有必要的机制来帮助折叠和促进复合物的形成,并试图通过共同表达这些蛋白质来产生人Hsp90β,人Cdc37和人Cdk4激酶之间的复合物, S上。酵母。为了获得三种蛋白质的化学计量表达,我们利用病毒2A肽,其允许三个蛋白质在一个mRNA上转录,随后在翻译阶段切割。该系统已被用于人类细胞系和兔网状细胞(Kim等人,2011; ...

Serial Immunoprecipitation of 3xFLAG/V5-tagged Yeast Proteins to Identify Specific Interactions with Chaperone Proteins
Author:
Date:
2017-06-20
[Abstract]  This method was generated to isolate high affinity protein complexes from yeast lysate by performing serial affinity purification of doubly tagged 3xFLAG/V5 proteins. First, the bait protein of interest is immunoprecipitated by anti-FLAG-conjugated magnetic beads and gently eluted by 3xFLAG antigen peptide. Next, the bait protein is recaptured from the first eluate by anti-V5-conjugated magnetic beads and eluted with ionic detergent. In this manner, the majority of abundant, nonspecific proteins remain either bound to the first beads or in the first eluate, allowing pure, high affinity complexes to be obtained. This approach can be used to show specific interactions with notoriously ‘sticky’ chaperone proteins. [摘要]  产生这种方法通过双重标记的3xFLAG / V5蛋白的连续亲和纯化来分离来自酵母裂解物的高亲和力蛋白质复合物。 首先,感兴趣的诱饵蛋白通过抗FLAG缀合的磁珠免疫沉淀,并用3xFLAG抗原肽轻轻洗脱。 接下来,通过抗V5共轭磁珠从第一洗脱液中回收诱饵蛋白质并用离子型洗涤剂洗脱。 以这种方式,大多数丰富的非特异性蛋白质仍然与第一珠粒或第一洗脱液结合,从而获得纯的高亲和性复合物。 这种方法可用于显示与臭名昭着的“粘滞”伴侣蛋白的特定相互作用。
【背景】通过质谱(IP / MS)进行免疫沉淀是鉴定与特定目标诱饵蛋白质的蛋白质 - 蛋白质相互作用的无偏见方法。虽然这种方法被有效地应用于鉴定蛋白质相互作用网络,但是它被假阳性困扰 - ...

In vitro Reconstitution Assay of miRNA Biogenesis by Arabidopsis DCL1
Author:
Date:
2015-04-20
[Abstract]  microRNAs (miRNAs) are small non-coding RNAs, regulating most if not all, biological processes in eukaryotic organisms. miRNAs are initially processed from primary transcripts (pri-miRNAs) to produce miRNA precursors (pre-miRNAs), that are further processed into miRNA and its complementary strands (miRNA/*). In Arabidopsis, and possibly other plants, the processing from pri-miRNAs to pre-miRNAs and from pre-miRNAs to miRNA/* are both implemented through Dicer-like 1 (DCL1) complexes. Recently, we demonstrated isolation of DCL1 complexes of unprecedented quality from in planta. We further successfully reconstituted DCL1 cleavage assays in vitro that were able to fully recapitulate in vivo miRNA biogenesis. Here we provide a detailed protocol of DCL1 ... [摘要]  微小RNA(miRNA)是小的非编码RNA,其调节真核生物中的大多数(如果不是全部)生物过程。 miRNA最初从初级转录物(pri-miRNA)加工以产生miRNA前体(前-miRNA),其进一步加工成miRNA及其互补链(miRNA/*)。在拟南芥和可能的其他植物中,从pri-miRNA到pre-miRNA和从pre-miRNA到miRNA/*的加工都通过Dicer样1(DCL1)复合物实现。最近,我们证明了从植物中以前的质量的DCL1复合物的分离。我们进一步成功地重建了能够完全重现体内 miRNA生物发生的DCL1切割测定。在这里我们提供DCL1重建测定的详细协议。该方案包括三个主要部分(图1):1)pri-miRNA和pre-miRNA转录物的制备(方法A-C); 2)通过免疫沉淀(IP)纯化来自本塞姆氏烟草(本塞姆氏烟草)的重组拟南芥DCL1机器(程序D和E);和3)使用分离的DCL1复合物(步骤F)在放射性同位素标记的pri-miRNA或pre-miRNA的体外处理。这是我们的愿望,协议是RNAi社区研究机械问题或开发RNA沉默技术的强大工具。

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