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TWEEN® 20

TWEEN ® 20

Company: Sigma-Aldrich
Catalog#: P1379
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Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay
Author:
Date:
2018-01-05
[Abstract]  This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in E. coli as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules out nucleic acid-mediated indirect interaction. Then, a second immunoprecipitation step is performed using the purified putative partner protein. Co-immunoprecipitated proteins can be detected either by Coomassie Blue staining and/or Western blotting (WB) if a specific antibody is available. Moreover, many DNA/RNA binding proteins are highly electropositive, which can hinder WB under standard conditions, as has been shown in histones and ... [摘要]  该协议通过下拉共免疫沉淀分析两种DNA结合蛋白之间的直接相互作用。其中一种蛋白在E中过表达。如HA标记的重组蛋白和无细胞提取物在HA亲和树脂中免疫沉淀。用核酸酶处理细胞提取物以降解DNA和RNA,这排除了核酸介导的间接相互作用。然后,使用纯化的推定的配偶体蛋白进行第二次免疫沉淀步骤。如果特异性抗体可用,可以通过考马斯蓝染色和/或Western印迹(WB)检测免疫共沉淀蛋白质。此外,许多DNA / RNA结合蛋白具有高度正电性,在标准条件下可阻碍WB,正如组蛋白和组蛋白样蛋白所示。在这种情况下,我们表明,假定的合作伙伴的高等电点导致转移不良。提示麻烦WB提供高正电荷DNA结合蛋白的转移。


【背景】共免疫沉淀是分析蛋白质 - 蛋白质相互作用(PPI)的常用方法。许多共免疫沉淀方案使用细菌表达的蛋白质。然而,细胞提取物的使用不排除由第三种蛋白介导的间接相互作用,或者在DNA / RNA结合蛋白的情况下介导核酸。

乙型病毒Bam35(B35TP)的末端蛋白含有保守的酪氨酸194,其提供OH基团以在蛋白质引发的DNA复制期间锚定病毒基因组的第一个5'-dTMP。此外,B35TP具有很强的DNA结合能力,与许多DNA结合蛋白一样,它具有非常高的等电点(约10.6),这影响其体外稳定性和功能(Berjón-Otero 等),2016)。 ...

MicroScale Thermophoresis as a Tool to Study Protein-peptide Interactions in the Context of Large Eukaryotic Protein Complexes
Author:
Date:
2017-12-05
[Abstract]  Protein-peptide interactions are part of many physiological processes, for example, epigenetics where peptide regions of histone complexes are crucial for regulation of chromatin structure. Short peptides are often also used as alternatives to small molecule drugs to target protein complexes. Studying the interactions between proteins and peptides is thus an important task in systems biology, cell biology, biochemistry, and drug design. However, this task is often hampered by the drawbacks of classical biophysical methods for analysis of molecular interactions like surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC), which require immobilization of the interaction partners or very high sample concentrations. MicroScale Thermophoresis (MST) is an innovative method ... [摘要]  蛋白质 - 肽相互作用是许多生理过程的一部分,例如表观遗传学,其中组蛋白复合物的肽区域对于染色质结构的调节是至关重要的。短肽通常也被用作小分子药物靶向蛋白质复合物的替代物。研究蛋白质和肽之间的相互作用是系统生物学,细胞生物学,生物化学和药物设计中的重要任务。然而,这一任务往往受到经典生物物理学方法分析分子间相互作用的缺陷的困扰,例如表面等离子体共振(SPR)或等温滴定量热法(ITC),其需要固定相互作用配偶体或非常高的样品浓度。 MicroScale热泳(MST)是一种创新的方法,可以确定分子间相互作用的重要参数,如解离常数,化学计量和热力学。而且,它可以快速准确地进行,可以自由选择缓冲液或生物液体,不需要固定样品,样品消耗也非常少。这里我们详细描述了两个MST测定法,其分析(i)真核RNA聚合酶II的某些肽段与真核转录延伸复合物的蛋白质亚基之间的相互作用和(ii)N-末端组蛋白尾肽与表观遗传学之间的相互作用读者蛋白质。这些实验表明,MST能够表征蛋白质 - 肽相互作用,这些相互作用仅由肽的微小变化触发,例如,在特定的丝氨酸残基处仅有一个磷酸化。

【背景】生物学背景:蛋白质 - ...

An Affinity-directed Protein Missile (AdPROM) System for Targeted Destruction of Endogenous Proteins
Author:
Date:
2017-11-20
[Abstract]  We recently reported an Affinity-directed PROtein Missile (AdPROM) system for the targeted proteolysis of endogenous proteins of interest (POI) (Fulcher et al., 2016 and 2017). AdPROM consists of the Von Hippel Lindau (VHL) protein, a Cullin 2 E3 ligase substrate receptor (Bosu and Kipreos, 2008), conjugated to a high affinity polypeptide binder (such as a camelid nanobody) that recognises the target protein in cells. When introduced in cells, the target protein is recruited to the CUL2 E3 ubiquitin ligase complex for ubiquitin-mediated proteasomal degradation. For target protein recruitment, we have utilised both camelid-derived VHH domain nanobodies as well as synthetic polypeptide monobodies based on the human type III fibronectin domain (Sha et al., 2013; Fridy et ... [摘要]  我们最近报道了一种针对内源性感兴趣蛋白(POI)的靶向蛋白水解的亲和指导PROtein导弹(AdPROM)系统(Fulcher等人,2016和2017)。 AdPROM由Von Hippel Lindau(VHL)蛋白组成,Cullin 2 E3连接酶底物受体(Bosu and Kipreos,2008),与识别细胞中靶蛋白的高亲和力多肽结合剂(如骆驼科纳米抗体)缀合。当在细胞中引入时,靶蛋白质被招募到CUL2 E3泛素连接酶复合体用于泛素介导的蛋白酶体降解。对于靶蛋白的募集,我们使用了基于人类III型纤连蛋白结构域的骆驼科动物来源的VHH结构域纳米抗体以及合成多肽单体(Sharm等人,2013; Fridy等人。,2014; Schmidt et al。,2016)。在此协议中,我们描述了生成AdPROM构建体及其在人细胞系中用于靶蛋白质破坏的详细方法。 AdPROM允许对POI进行功能表征,并且其目标蛋白质破坏的效率克服了RNA干扰方法的许多局限性,这些方法需要长时间的治疗并与脱靶效应相关联,而CRISPR / Cas9基因编辑并不总是可行的。
【背景】该协议使人们能够在哺乳动物细胞系中设计,构建和表达AdPROM VHL-nano ...

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