| Extraction and Quantification of Polyphosphate in the Budding Yeast Saccharomyces cerevisiae
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Author:
Date:
2016-07-20
[Abstract] Inorganic polyphosphate (polyP) is a linear polymer present in both prokaryotic and eukaryotic organisms and made from three to hundreds of orthophosphate residues linked by phosphoanhydride bonds. The biological role of this molecule goes beyond serving as Pi store or energy source to replace ATP. For instance, in yeast polyP levels have been related to stress adaptation and this molecule has been shown to be the substrate for polyphosphorylation of proteins. Here we describe two different methods to purify polyP from the yeast Saccharomyces cerevisiae and the subsequent protocol to quantify polyP levels by spectrophotometrically measuring the Pi generated upon enzymatic hydrolysis of purified polyP. It must be noted that the purification protocol used greatly influences the ...
[摘要] 无机多磷酸盐（polyP）是存在于原核和真核生物中的线性聚合物，由通过磷酸酐键连接的三至数百个正磷酸盐残基制成。 这种分子的生物学作用超越了作为P 1储存或能量源以代替ATP。 例如，在酵母中，polyP水平与应激适应相关，并且该分子已经显示为蛋白质多磷酸化的底物。 在这里，我们描述了从酵母酿酒酵母中纯化polyP的两种不同方法和随后的通过分光光度法测量在酶水解纯化的polyP时产生的Pi来定量polyP水平的方案。 必须注意的是，所使用的纯化方案极大地影响所获得的polyP值。
图1. polyP的酶水解
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| Protein Extraction from Drosophila Embryos and Ovaries
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Author:
Date:
2015-05-05
[Abstract] Here we provide the description of protocols to efficiently obtain protein extracts from embryos and ovaries of Drosophila melanogaster. These protocols are routinely applied in our laboratory and are based on two techniques: either embryos or ovaries are homogenized using a pestle and then the soluble proteins separated by centrifugation, or embryos are individually lysed by needle manipulation. The latter technique allows the use of small embryo numbers and the selection of specific developmental stages (Guilgur et al., 2014).
[摘要] FLP / FRT系统是基于将重组酶（翻转酶-FLP）靶向指定为翻转酶识别靶标（FRT）位点的特定DNA区域的定点重组技术。最初在酿酒酵母中鉴定，酵母FLP酶及其FRT重组靶点成功转移到果蝇中的每个主要染色体臂（Golic和Lindquist，1989）。这提供了以受控方式在发育过程中在体内介导有丝分裂重组的能力[在Theodosiou和Xu（1998）中修订]。有丝分裂重组事件的受控诱导通常通过在热休克（hs）启动子的控制下表达FLP来进行。这允许在特定发育时间窗口表达高FLP水平。携带这些遗传标记的FLP / FRT染色体的菌株大大增强了我们在种系和体细胞果蝇组织中研究基因功能的能力。在这里我们描述两种不同的方案：一种用于诱导和鉴定卵巢中的纯合突变体克隆，另一种用于产生雌性种系突变体，用于分析母体对胚胎发生的影响。
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| Purification of Starch Granules from Arabidopsis Leaves and Determination of Granule-Bound Starch Synthase Activity
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Author:
Date:
2014-12-05
[Abstract] Starch constitutes the most important carbon reserve in plants and is composed of branched amylopectin and linear amylose. The latter is synthesized exclusively by the Granule-Bound Starch Synthase (GBSS, EC 2.4.1.21). Here we report a readily reproducible, specific and highly sensitive protocol, which includes the isolation of intact starch granules from Arabidopsis thaliana leaves and the subsequent determination of GBSS activity. We have applied this method to study GBSS activity in diurnal cycles in vegetative growth and during the photoperiodic transition to flowering in Arabidopsis (Tenorio et al., 2003; Ortiz-Marchena et al., 2014).
[摘要] 淀粉构成植物中最重要的碳储量，由支链支链淀粉和线性直链淀粉组成。 后者仅由颗粒结合的淀粉合酶（GBSS，EC 2.4.1.21）合成。 在这里我们报告一个容易重现，具体和高度敏感的协议，其中包括完整的淀粉颗粒从拟南芥叶中分离和随后的GBSS活动的确定。 我们已经应用这种方法来研究营养生长的日变化周期中和在拟南芥中的光周期过渡到开花期间的GBSS活性（Tenorio等人，2003; Ortiz-Marchena& em> et al。，2014）。
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