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Rifampicin

利福平

Company: Sigma-Aldrich
Catalog#: R3501
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Quantification of Plant Cell Death by Electrolyte Leakage Assay
Author:
Date:
2018-03-05
[Abstract]  We describe a protocol to measure the electrolyte leakage from plant tissues, resulting from loss of cell membrane integrity, which is a common definition of cell death. This simple protocol is designed to measure the electrolyte leakage from a tissue sample over a time course, so that the extent of cell death in the tissue can be monitored dynamically. In addition, it is easy to handle many tissue samples in parallel, which allows a high level of biological replication. Although the protocol is exemplified by cell death in Arabidopsis in response to pathogen challenge, it is easily applicable to other types of plant cell death. [摘要]  我们描述了测量植物组织中电解质渗漏的方案,其由细胞膜完整性的丧失导致,这是细胞死亡的常见定义。 这个简单的方案设计用于测量组织样本在一段时间内的电解质泄漏,从而可以动态监测组织中细胞死亡的程度。 另外,平行处理许多组织样品很容易,这允许高水平的生物复制。 尽管该方案以响应病原体攻击的拟南芥中的细胞死亡为例,但它很容易应用于其他类型的植物细胞死亡。

【背景】当细胞死亡并丧失细胞膜的完整性时,电解质如K +离子就会从细胞中渗出。因此,我们可以使用组织中泄漏的电解质的量作为组织中细胞死亡程度的代表。量化从组织泄漏的电解质的简单方法是测量含有将死细胞的组织的水的电解电导率的增加。这种电解质渗漏测定法已经应用于植物组织,以评估响应于生物和非生物胁迫而死亡的细胞的相对数量,所述细胞例如病原体攻击,昆虫食草,伤口,UV辐射,氧化应激,盐度,干旱,寒冷和热压力(Demidchik et al。,2014)。

最初的方法被设计成测量含有植物组织的水浴溶液在煮沸之前和之后的电导率,其中煮沸后的电导率被用于使组织尺寸差异标准化(Whitlow等人,1992年)。在这里我们描述了一个程序,通过在多个时间点测量叶片浮在12孔板上的水的电解电导率来动态监测叶盘中的电解质渗漏。可以合理地假设来自相同大小的组织样品的电解质的总量,例如从类似发育阶段的叶子冲出的相同面积的圆盘是相当的,并且没有必要测量电解质电导率之后煮沸组织。我们用细菌病原体,即丁香假单胞菌 ...

Use of Geminivirus for Delivery of CRISPR/Cas9 Components to Tobacco by Agro-infiltration
Author:
Date:
2017-04-05
[Abstract]  CRISPR/Cas9 system is a recently developed genome editing tool, and its power has been demonstrated in many organisms, including some plant species (Wang et al., 2016). In eukaryotes, the Cas9/gRNA complexes target genome sites specifically and cleave them to produce double-strand breaks (DSBs), which can be repaired by non-homologous end joining (NHEJ) pathway (Wang et al., 2016). Since NHEJ is error prone, mutations are thus generated. In plants, delivery of genome editing reagents is still challenging. In this protocol, we detail the procedure of a virus-based gRNA delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE). This method offers a rapid and efficient way to deliver gRNA into plant cells, especially for those that are recalcitrant to ... [摘要]  CRISPR / Cas9系统是最近开发的基因组编辑工具,其功能已被证实在许多生物体中,包括一些植物物种(Wang等人,2016)。 在真核生物中,Cas9 / gRNA复合物特异性地靶向基因组位点并切割它们以产生双链断裂(DSB),其可以通过非同源末端连接(NHEJ)途径修复(Wang等人, 。,2016)。 由于NHEJ易出错,因此产生突变。 在植物中,基因组编辑试剂的递送仍然是挑战性的。 在本协议中,我们详细介绍了CRISPR / Cas9介导的植物基因组编辑(VIGE)的基于病毒的gRNA传递系统的过程。 该方法提供了将gRNA递送到植物细胞中的快速且有效的方式,特别是对于那些难以转基因农杆菌的方法。

已经报道了基于病毒的基因组编辑技术使用解构DNA病毒和RNA病毒(Baltes等人,2014; Ali等人,2015)。 最近,我们使用了一种完整的双因素病毒 - 卷心菜叶卷曲病毒(CaLCuV)(一种感染广西芥菜科的成员,包括花椰菜的二分酵母病毒),用于高效率 第一次(Yin等人,2015年),其主机之一的基因组编辑(Nicotiana benhamiana)首次进行基因组编辑。

Cell-based Assays to Monitor AID Activity
Author:
Date:
2016-02-05
[Abstract]  The enzyme Activation induced deaminase (AID) underpins antibody affinity maturation and isotype switching through its mutagenic activity of deaminating deoxycytidine to deoxyuridine in DNA. Subsequent processing of the deoxyuridine initiates the processes of somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. Structure-function analysis of AID requires sensitive and biologically relevant methods to measure its various activities. Here we describe simple but effective methods to measure 1) the ability of AID to mutate the Escherichia coli genome, which provides an indication of its catalytic activity; 2) the capacity of AID to perform SHM by complementing a derivative of the DT40 chicken B cell line; 3) the ability of AID to perform CSR by complementing ... [摘要]  酶活化诱导的脱氨酶(AID)通过其将脱氧胞苷脱氨基到DNA中的脱氧尿苷的诱变活性来支持抗体亲和力成熟和同种型转换。脱氧尿苷的后续加工引发B细胞中体细胞超突变(SHM)和类型转换重组(CSR)的过程。 AID的结构功能分析需要灵敏和生物相关的方法来测量其各种活动。在这里我们描述简单但有效的方法来测量1)AID突变大肠杆菌基因组的能力,其提供其催化活性的指示; 2)AID通过补充DT40鸡B细胞系的衍生物来进行SHM的能力; 3)AID通过补充AID缺乏的原代小鼠B细胞来进行CSR的能力。三种方法的组合,伴随着AID亚细胞定位和蛋白质表达水平和稳定性的必要分析作为对照,允许AID的详细结构功能研究。

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