Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay
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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)。 ...
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The Application of Quercetin to Study the Effect of Hsp70 Silencing on Plant Virus Infection in Nicotiana benthamiana Plants
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Author:
Date:
2015-12-05
[Abstract] Pepino mosaic virus (PepMV) is a mechanically-transmitted pathogen affecting tomato plants worldwide. Like with other plant viruses (Verchot, 2012), the heat shock cognate protein 70 homolog (Hsc70) was identified as an interactor of the PepMV coat protein (CP) (Mathioudakis et al., 2012). Here, we describe a pharmacological approach to silence Hsp70 in plants using quercetin (Mathioudakis et al., 2014), an Hsp70 protein expression flavonoid inhibitor (Hosokawa et al., 1990; Manwell and Heikkila 2007). In the case of Hsp70, this methodology represents a faster and easier approach than silencing of Hsp70 by reverse genetics assays, such as VIGS methodology. Fully expanded leaves of 2 to 3 weeks old Nicotiana benthamiana plants were ...
[摘要] Pepino花叶病毒( PepMV )是一种机械传播的病原体,影响全世界的番茄植株。与其他植物病毒一样(Verchot,2012),热休克同源蛋白70同源物(Hsc70)被鉴定为PepMV外壳蛋白(CP)的相互作用物(Mathioudakis等人,2012)。在这里,我们描述了使用槲皮素使沉默Hsp70在植物中的药理学方法(Mathioudakis等人,2014),Hsp70蛋白质表达类黄酮抑制剂(Hosokawa等人,1990 ; Manwell和Heikkila 2007)。在Hsp70的情况下,该方法表示比通过反向遗传学测定(例如VIGS方法)沉默Hsp70更快和更容易的方法。使用注射器用槲皮素(溶解于DMSO中)或DMSO(对照植物)浸润2至3周龄的烟草烟草的完全膨胀的叶子。用PepMV病毒接种物机械接种植物。在接种后4天通过免疫印迹分析在局部叶子上分析Hsp70和PepMV的积累。
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