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Author:
Date:
2015-04-05
[Abstract] For monitoring the cellular pH in plants, engineered green fluorescent protein (GFP) has been used to indicate cellular pH. Pt-GFP is a pH-reporter protein which has been used ratiometrically in the double excitation mode, according to the fluorescence properties described for Pt-GFP (Schulte et al., 2006), and depending on our previous experiments, we present here a little and efficient protocol for monitoring the cytosolic pH value. In this protocol, root hair cellular pH was monitored in the PtGFP reporter lines. Cellular pH can be obtained according to the calibration curve which was performed in situ by using a series pH buffer.
[摘要] 为了监测植物中的细胞pH，使用工程绿色荧光蛋白（GFP）来指示细胞pH。 根据Pt-GFP（Schulte等人，2006）所述的荧光性质，Pt-GFP是pH报道蛋白，其已经在双激发模式中按比例计量使用，并且取决于我们 以前的实验，我们在这里提出一个有效的协议，用于监测胞浆pH值。 在该协议中，在PtGFP报告系中监测根毛细胞pH。 可以根据通过使用系列pH缓冲液原位进行的校准曲线获得细胞pH。
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Author:
Date:
2013-12-20
[Abstract] Membrane preparation has been widely used for characterization the membrane proteins. Membrane fractions can be separated by a combination of differential and density-gradient centrifugation techniques (Hodges et al., 1972; Leonard and Vanderwoude, 1976). Here we firstly describe a method to isolate total microsomal fractions including plasma membrane, intracellular vesicles, Golgi membranes, endoplasma reticulum, and tonoplast (vacuolar membrane) from 5-7 days old seedlings, which is often analyzed for auxin transporters in Arabidopsis (Leonard and Vanderwoude, 1976; Titapiwatanakun, et al., 2009; Yang et al., 2013; Blakeslee et al., 2007). After homogenization, plant debris including cell walls, chloroplasts and nucleus were removed by low ...
[摘要] 膜制备已广泛用于表征膜蛋白。膜级分可以通过差示和密度梯度离心技术的组合来分离（Hodges等人，1972; Leonard和Vanderwoude，1976）。在这里我们首先描述一种从5-7天龄幼苗中分离总微粒体级分包括质膜，胞内囊泡，高尔基体膜，内质网和液泡膜（液泡膜）的方法，其通常在拟南芥中分析生长素转运蛋白（Leonard和Vanderwoude，1976; Titapiwatanakun，，2009; Yang等人，2013; Blakeslee等人 >，2007）。均化后，通过低速离心（8,000×g）除去包括细胞壁，叶绿体和细胞核的植物碎片，然后通过高速离心（10,000×g/ml）沉淀总微粒体膜， ）并与可溶性级分分离。我们第二次描述了通过离心在蔗糖密度梯度系统中根据大小或密度分离微粒体部分的方法。使用20％-55％（1.09-1.26g cm -3）的线性蔗糖梯度分离具有不同密度的膜：tonoplast，1.10-1.12cm 3 - ，高尔基体膜，1.12-1.15cm 3，粗糙的内质网1.15-1.17cm 3，类囊体，1.16-1.18cm 3 - ，质膜，1.14-1.17g cm -3 - 和线粒体膜，1.18-1.20cm -3（Leonard和Vanderwoude，1976; Larsson等人， ，1987; Briskin and ...
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