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DL-Dithiothreitol

DL-二硫苏糖醇

Company: Sigma-Aldrich
Catalog#: 43819
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Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays
Author:
Date:
2016-12-05
[Abstract]  We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to mature for defined periods inside cells before extraction for in vitro motility. These assays allow us to probe how lipids on the phagosome membrane recruit and organize motors, and also measure the motion and force generation resulting from underlying lipid-motor interactions. This provides a unique opportunity to interrogate native-like organelles using biophysical and biochemical assays, and understand the role of motor proteins in phagosome maturation ... [摘要]  芽顶端分生组织(SAM)是通过细胞分裂不断更新自身的细胞的集合,并且还向新发育的器官提供细胞。已知CLAVATA(CLV)3肽调节转录因子WUSCHEL(WUS)以保持未分化细胞的数量恒定并维持SAM的大小。在非细胞自主信号级联中的CLV3和WUS的交互反馈控制确定SAM中的干细胞命运(多能性的维持,或者,分化成子细胞)。 Ca 2 + 是在许多信号传导途径中起重要作用的第二信使。连接CLV3结合其受体和WUS表达的信号系统没有很好地描绘。我们显示Ca 2 + 参与SAM大小的CLV3调节。我们使用的方法之一是测量SAM的大小。在这里,我们提供了一个详细的协议,如何用Nomarski显微镜测量拟南芥SAM大小。将代表SAM的最大"面"的二维圆顶的面积用作SAM大小的代表。在存在和不存在Ca 2+通道阻断剂Gd 3+和sLV 3肽的情况下对野生型(WT)拟南芥进行研究。 ,以及缺乏功能性CLV3( clv3 )或编码Ca 2+ 2+导电离子通道的基因('dnd1 ')的基因型。 关键字: 拟南芥,射击顶端分生组织,苗发育,细胞信号,幼苗 > Nomarski显微镜广泛用于研究拟南芥SAM大小。用于SAM观察的其他显微技术是耗时的,并且需要将树脂嵌入树脂中,然后切片或甚至更复杂的显微镜。 ...

Protein Extraction from Drosophila Embryos and Ovaries
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。

Gradient Flotation Centrifugation of Chloroplast Membranes
Author:
Date:
2014-09-05
[Abstract]  Plastoglobules are lipoprotein particles physically attached to thylakoids in chloroplasts (Kessler et al., 1999). They are mainly composed of polar lipid, neutral lipids, and proteins (Vidi et al., 2006). Here we used simple sucrose gradient flotation centrifugation method to purify the plastoglobules from total chloroplast membranes (Vidi et al., 2007, Shanmugabalaji et al., 2013). [摘要]  塑性球蛋白是物理附着于叶绿体中类囊体的脂蛋白颗粒(Kessler等人,1999)。 它们主要由极性脂质,中性脂质和蛋白质组成(Vidi等人,2006)。 在这里,我们使用简单的蔗糖梯度浮选离心法从总叶绿体膜中纯化质体球(Vidi等人,2007,Shanmugabalaji等人,2013)。

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