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Bovine Serum Albumin

牛血清白蛋白

Company: Sigma-Aldrich
Catalog#: A4503
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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)。 ...

Immunostaining of Formaldehyde-fixed Metaphase Chromosome from Untreated and Aphidicolin-treated DT40 Cells
Author:
Date:
2017-05-05
[Abstract]  During mitosis chromosomes are condensed into dense X-shaped structures that allow for microscopic determination of karyotype as well as inspection of chromosome morphology.

This protocol describes a method to perform immunostaining of formaldehyde-fixed metaphase chromosomes from the avian cell line DT40. It was developed to characterize the localization of YFP-tagged TopBP1 on mitotic chromosomes and specifically determine the percentage of TopBP1 foci that formed on breaks/gaps as well as ends of individual metaphase macrochromosomes (Pedersen et al., 2015). For this purpose immunostaining of YFP was applied. However, the protocol may be optimized for other cell lines or epitopes.
[摘要]  在有丝分裂期间,染色体被浓缩成致密X型结构,允许显微检测染色体核型和检查染色体形态。
该方案描述了从禽细胞系DT40进行甲醛固定的中期染色体的免疫染色的方法。 它被开发用于表征YFP标记的TopBP1在有丝分裂染色体上的定位,并且具体确定形成于断裂/间隙以及单个中期大染色体末端的TopBP1灶的百分比(Pedersen等,2015)。 为此,应用YFP的免疫染色。 然而,该协议可以针对其他细胞系或表位进行优化。
【背景】染色中期染色体的显微镜分析是经典的细胞遗传学技术,广泛用于研究和诊断。基本原理包括通过锭子不稳定试剂如谷氨酸诱导中期细胞周期停滞,这将触发主轴装配检查点,从而阻止细胞在中期。这用于富集浓缩染色体的细胞。随后将细胞在低渗溶液中进行溶胀,然后在显微镜载玻片上铺展有丝分裂细胞。最终结果是来自单细胞的显微镜检测染色体,便于染色体核型分析以及个体染色体的研究。传统上,肿胀的细胞用甲醇和乙酸(3:1)固定,然后铺展在载玻片上(Hungerford,1965; Ronne等,1979)。
这里描述的方法使用甲醛而不是甲醇进行固定。这可以用于随后用与甲醇固定不相容的抗体染色。该方案针对鸡DT40细胞的中期传播进行了优化,YFP标记的TopBP1在中期大染色体上的免疫染色(Pedersen等,2015)。 ...

Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery
Author:
Date:
2017-04-05
[Abstract]  The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair mechanism. However, in presence of a DNA repair template, Homology-Directed Repair (HDR) can occur leading to precise repair of the lesion site. This last process can be exploited to enable precise knock-in changes by introducing the desired genomic alteration on the repair template. In this protocol we describe the delivery of long repair templates (> 200 nucleotides) using recombinant Adeno Associated Virus (rAAV) for CRISPR-Cas9-based knock-in of a ... [摘要]  可编程集群定期间隔短回归度(CRISPR)相关核酸酶9(Cas9)技术通过提供在所需位置切割基因组的有效方式,彻底改变了基因组编辑(Ledford,2015)。 在哺乳动物细胞中,DNA损伤触发易发生非同源末端连接(NHEJ)DNA修复机制。 然而,在DNA修复模板的存在下,可以发生同源性定向修复(HDR),导致病变部位的精确修复。 可以利用最后的方法,通过在修复模板上引入所需的基因组改变来实现精确的敲入变化。 在本协议中,我们描述了使用重组腺相关病毒(rAAV)在人细胞系中进行基于CRISPR-Cas9的C-末端标签序列敲入的长修复模板(> 200个核苷酸)的递送。

尽管有关CRISPR-Cas9产生的敲门模型系统的大量报告,敲门砖报告仍然落后。由于许多应用,产生敲入细胞系仍然是基因组编辑的明显目标。敲入改变的引入通常依赖于修复模板DNA的存在,并且在位点特异性双链(ds)DNA断裂被引入接近改变位点的基因组中后,HDR修复机制的激活。不同的模板可以传送到修复机器,范围从含有广泛同源区域和可选选择盒的经典线性化载体到约200个核苷酸的单链(ss)DNA寡核苷酸(Chen等人, ...

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