| Semi-thin Sectioning, Light and Fluorescence Microscopy of Floral Bud to Study Microspore Development in Arabidopsis
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Author:
Date:
2016-03-05
[Abstract] Pollen grains are male gametophytes produced within the pollen sacs of the anthers of the flower. Recent genetic studies have revealed several components involved in microspore development (Borg et al., 2009; Berger and Twell, 2011), and yet many components controlling microspore development remain to be identified. Semi-thin sectioning of anthers and light and fluorescence microscopy of floral bud (Kim et al., 2015) are the initial key experiments to characterize Arabidopsis mutants and transgenic plants for understanding the roles of new genetic components during ...
[摘要] 花粉粒是在花的花药的花粉囊内产生的雄性配子体。 最近的遗传研究揭示了涉及小孢子发育的几种组分（Borg等人，2009; Berger和Twell，2011），然而仍有许多控制小孢子发育的组分被鉴定。 花药的半薄切片和花芽的光和荧光显微镜检查（Kim等人，2015）是表征拟南芥突变体和转基因植物的初始关键实验 在小孢子发育过程中新遗传组分的作用。 在这里，我们描述了拟南芥花药半花瓣和光和荧光显微镜的半薄切片的协议。
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| Cell-based Assays to Monitor AID Activity
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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 ...
[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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| Protein Extraction, Acid Phosphatase Activity Assays, and Determination of Soluble Protein Concentration
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Author:
Date:
2013-09-05
[Abstract] Acid phosphatases (APases) catalyze the hydrolysis of inorganic phosphate (Pi) from a broad range of Pi-monoesters with an acidic pH optimum. The liberated Pi is reassimilated into cellular metabolism via mitochondrial or chloroplastic ATP synthases of respiration or photosynthesis, respectively. Eukaryotic APases exist as a wide variety of tissue- and/or cellular compartment-specific isozymes that display marked differences in their physical and kinetic properties. Increases in intracellular (vacuolar) and secreted APase activities are useful biochemical markers of plant nutritional Pi ...
[摘要] 酸性磷酸酶（APase）催化来自具有酸性pH最佳值的宽范围的P 1 - 单酯的无机磷酸盐（P 1）的水解。 释放的Pi分别通过呼吸或光合作用的线粒体或叶绿体ATP合酶再吸收到细胞代谢中。 真核APase作为多种组织和/或细胞区室特异性同工酶存在，其在它们的物理和动力学性质上显示出显着的差异。 细胞内（液泡）和分泌的APase活性的增加是植物营养缺乏的有用的生物化学标记物。 提出了蛋白质提取，APase活性测定和来自植物组织或细胞悬浮培养物的可溶性蛋白质浓度的测定方案。
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