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OptimaTM TLX Centrifuge

Optima TM TLX超速离心机,60Hz,110V

Company: Beckman Coulter
Catalog#: OptimaTM TLX
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Isolation and Quantification of Plant Extracellular Vesicles
Author:
Date:
2017-09-05
[Abstract]  Extracellular vesicles (EVs) play an important role in intercellular communication by transporting proteins and RNA. While plant cells secrete EVs, they have only recently been isolated and questions regarding their biogenesis, release, uptake and function remain unanswered. Here, we present a detailed protocol for isolating EVs from the apoplastic wash of Arabidopsis thaliana leaves. The isolated EVs can be quantified using a fluorometric dye to assess total membrane content. [摘要]  细胞外囊泡(EVs)通过传递蛋白质和RNA在细胞间通讯中发挥重要作用。 虽然植物细胞分泌电动汽车,但是它们最近才被孤立,并且关于它们的生物发生,释放,摄取和功能的问题仍然没有得到回答。 在这里,我们提出了一个详细的方案,用于从拟南芥叶片的脱水洗涤中分离EV。 可以使用荧光染料定量分离的EV,以评估总膜含量。
【背景】细胞外囊泡(EVs)是介导蛋白质,脂质和遗传物质的细胞与细胞转移的膜结合结构。由于哺乳动物EVs转运RNA和调节免疫反应的能力,对哺乳动物EV的兴趣已经增长。哺乳动物EV通常被分离用于从培养细胞的培养基中研究,以及生物流体的增长列表(Colombo等,2014)。植物电动车也被认为在免疫反应中起作用,但比较缺乏(An et al。,2007; Davis et al。,2016)。这在很大程度上归因于没有孤立的方法。
  虽然植物EVs自1967年以来一直被观察到,使用透射电子显微镜,但直到2009年才开发出分离方法(Halperin和Jensen,1967)。 ...

Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells
Author:
Date:
2017-09-05
[Abstract]  The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms underlying the P2X7 specific properties remain poorly understood, partly because purification of this eukaryotic membrane protein has been challenging. Here we describe a detailed protocol for expressing and purifying a mammalian P2X7 receptor using an insect cell-baculovirus system. The P2X7 receptor is expressed in Sf9 insect cells as a GFP fusion protein and solubilized with a buffer containing Triton X-100 detergent. The P2X7-GFP fusion protein is ... [摘要]  P2X7受体是仅在真核生物中发现的胞外ATP门控离子通道(Bartlett等,2014)。由于其P2X受体之间的独特性质,例如大电导孔的形成,P2X7受体已经涉及破坏性疾病如慢性疼痛(North和Jarvis,2013)。然而,P2X7特异性属性的机制仍然知之甚少,部分原因是纯化这种真核膜蛋白是一个挑战。在这里,我们描述了使用昆虫细胞 - 杆状病毒系统表达和纯化哺乳动物P2X7受体的详细方案。 P2X7受体在作为GFP融合蛋白的Sf9昆虫细胞中表达,并用含有Triton X-100洗涤剂的缓冲液溶解。然后使用Strep-Tactin亲和层析在含有十二烷基麦芽糖苷的缓冲液中纯化P2X7-GFP融合蛋白。在通过凝血酶酶切割连接的GFP和Strep-标签后,使用大小排阻色谱分离P2X7受体。该方法通常从6L的Sf9培养物产生约2mg的纯化蛋白质。纯化的蛋白质可以用含有15%甘油的缓冲液在4℃下储存至少2个月,并用于各种功能和结构研究(Karasawa和Kawate,2016)。
【背景】P2X7受体是嘌呤能P2X受体家族的七种亚型之一,并且是广泛疾病如神经退行性疾病,癫痫和神经性疼痛的有希望的新型药物靶点(North和Jarvis,2013; Bhattacharya和Biber, ...

Measurement of ATP Hydrolytic Activity of Plasma Membrane H+-ATPase from Arabidopsis thaliana Leaves
Author:
Date:
2016-12-05
[Abstract]  Plant plasma membrane H+-ATPase, which is a P-type ATPase, couples ATP hydrolysis to H+ extrusion and thereby generates an electrochemical gradient across the plasma membrane. The proton gradient is necessary for secondary transport, cell elongation, and membrane potential maintenance. Here we describe a protocol for measurement of the ATP hydrolytic activity of the plasma membrane H+-ATPase from Arabidopsis thaliana leaves. [摘要]  作为P型ATP酶的植物质膜H sup + -ATPase将ATP水解耦合到H + +/- 挤出,从而在质膜上产生电化学梯度。质子梯度对于二次转运,细胞伸长和膜电位维持是必需的。这里我们描述用于测量来自拟南芥叶片的质膜H + -ATPase的ATP水解活性的方案。
关键词: strong> 拟南芥,ATP水解活性,原钒酸盐,P-型ATP酶,血浆膜H + -ATPase

质膜H + -ATPase活性的测定对于阐明其功能和调节机制是重要的。然而,有时难以确定质膜H sup + -ATP酶的ATP水解活性,因为植物细胞含有许多ATP水解酶。该协议是基于Uemura和Yoshida(1986)和Kinoshita等人的出版物开发的。 (1995)。我们使用KNO 3作为V型ATP酶的抑制剂,钼酸铵作为酸性磷酸酶的抑制剂,寡霉素作为F型ATP酶的抑制剂,NaF作为磷酸酶的抑制剂(Shimazaki和Kondo ,1987; Kinoshita等人,1995)。原钒酸盐抑制P型ATP酶,并且因此可以通过评估来自ATP水解的钒酸盐敏感性P 1释放来用于测量质膜H sup + -ATP酶的活性。释放的P 1与钼酸盐反应形成蓝色络合物,然后可以通过测量在750nm的吸收来量化。

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